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Endocrinology Vol. 141, No. 1 197-207
Copyright © 2000 by The Endocrine Society

ARTICLES

The Effect of the Endozepine Triakontatetraneuropeptide on Corticosteroid Secretion by the Frog Adrenal Gland Is Mediated by Activation of Adenylyl Cyclase and Calcium Influx through T-Type Calcium Channels1

Olivier Lesouhaitier, Magloire K. Kodjo2, Franck Cartier, Vincent Contesse, Laurent Yon, Catherine Delarue and Hubert Vaudry

European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Unité Affiliée au Centre National de la Recherche Scientifique, 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, Unité Affiliée au Centre National de la Recherche Scientifique, 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 recently found that, in the frog adrenal gland, endozepines are present in chromaffin cells and we have shown that the triakontatetraneuropeptide TTN is a potent stimulator of corticosteroid secretion in vitro. In the present study, we have investigated the transduction mechanisms mediating the corticotropic effect of TTN on adrenocortical cells. Incubation of adrenal explants with graded concentrations of TTN induced a dose-dependent increase in cAMP formation, but did not affect polyphosphoinositide metabolism. Pretreatment of adrenal cells with the protein kinase A inhibitor H-89 markedly reduced the stimulatory effect of TTN on corticosterone and aldosterone secretion by perifused cells, whereas the phospholipase C inhibitor U-73122 did not affect the TTN-evoked stimulation of corticosteroid output. Incubation of adrenal cells with cholera toxin abolished the stimulatory effect of TTN on steroid secretion. Administration of a brief pulse of TTN (10-6 M) in the vicinity of cultured adrenocortical cells induced a robust increase in the concentration of intracellular calcium ([Ca2+]i). Repeated pulses of TTN resulted in a gradual attenuation of the responses, indicating the existence of a desensitization phenomenon. Incubation of the cells with the T-type calcium channel blocker mibefradil significantly reduced the TTN-evoked [Ca2+]i increase, whereas the L-type calcium channel blocker nifedipine and the N-type calcium channel blocker {omega}-conotoxin GVIA had no effect. Incubation of adrenal cells with H-89 markedly reduced the stimulatory effect of TTN on [Ca2+]i. The involvement of calcium in steroid secretion induced by TTN has also been investigated. Administration of mibefradil significantly reduced the TTN-evoked stimulation of steroid production, whereas nifedipine was devoid of effect. Taken together, these data indicate that in frog adrenocortical cells, the endozepine TTN stimulates cAMP formation and calcium entry through T-type calcium channels. The effects of TTN on the adenylyl cyclase/protein kinase A pathway and calcium influx both contribute to the stimulatory action of the peptide on corticosteroid secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TERM ENDOZEPINES designates a family of regulatory peptides that have been initially isolated from brain extracts on the basis of their ability to displace benzodiazepines from their binding sites. All endozepines identified to date derive from an 86-amino acid polypeptide called diazepam binding inhibitor (DBI) (1). The sequence of DBI has been determined in several vertebrate species, including human and ox (2), pig (3), rat (4), duck (5), frog (6), and carp (7). The primary structure of DBI has been well conserved during evolution, suggesting that this polypeptide plays important biological functions. Proteolytic cleavage of DBI generates several biologically active peptides, including the triakontatetraneuropeptide (TTN) DBI-(17–50) (8, 9), the octadecaneuropeptide (ODN) DBI-(33–50) (10), and the triakontaseptaneuropeptide DBI-(39–75) (11).

Endozepines are widely distributed in the central nervous system and in various peripheral organs (3, 12, 13, 14, 15, 16, 17). Consistent with their broad distribution, endozepines have been found to exert a large array of biological activities, such as modulation of melanotropin release from pituitary cells (18), inhibition of glucose-induced insulin release from the pancreas (19), stimulation of cholecystokinin secretion from the intestine (20), and stimulation of steroid synthesis in brain tissue (21).

The occurrence of high concentrations of DBI messenger RNA and/or DBI-related peptides has been demonstrated in the rat adrenal gland (13), ovary (22), and testis (23) and in the mouse Y1 adrenocortical cell line (24). In adrenocortical and Leydig cells, endozepines act as intracrine factors that stimulate steroidogenesis via activation of a peripheral-type benzodiazepine receptor located on the outer mitochondrial membrane (24, 25, 26). In Leydig cells, DBI also acts as a paracrine and/or autocrine factor stimulating peripheral-type benzodiazepine receptor located on the plasma membrane (26).

We have recently found that in the frog adrenal gland, endozepines are expressed in both chromaffin cells and a population of mast-like cells called Stilling cells (27). We have also shown that TTN is a potent stimulator of corticosteroid secretion in vitro (28), suggesting that endozepines act as paracrine factors regulating the activity of frog adrenocortical cells. In the course of these studies it was found that the corticotropic action of TTN is not mediated by central- or peripheral-type benzodiazepine receptors (28). The aim of the present study was to investigate the transduction mechanisms mediating the stimulatory effect of TTN on frog adrenocortical cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male frogs (Rana ridibunda; 40–50 g BW) were obtained from a commercial supplier (Couétard, Saint-Hilaire de Riez, France). The animals were kept in glass tanks supplied with tap water in a temperature-controlled room (8 C) under an established photoperiod (lights on, 0600–1800 h) for at least 1 week before use. Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.

Reagents and test substances
Synthetic TTN and ODN (human sequences) were obtained from Neosystem (Strasbourg, France). Endothelin-1 (ET-1) was synthesized by solid phase methodology as previously described (29). Leibovitz culture medium (L15), collagenase (type IA), protease (from Bacillus polymyxa, type IX), 3-isobutyl-1-methylxanthine, thapsigargin, cholera toxin, pertussis toxin, nifedipine, and {omega}-conotoxin GVIA were purchased from Sigma (St. Louis, MO). Mibefradil (dihydrochloride) was provided by Hoffmann-La Roche (Basel, Switzerland). FBS, kanamycin solution (10,000 U/ml), and antibiotic-antimycotic solution (penicillin G sodium, 10,000 U/ml; streptomycin sulfate, 10,000 U/ml; amphotericin-B, 25 mg/ml) were obtained from Life Technologies, Inc. (Grand Island, NY). HEPES was purchased from Merck & Co., Inc. (Darmstadt, Germany). Myo-[3H]inositol (100 Ci/mmol), the cAMP RIA kit, [1,2,6,7-3H]corticosterone, and [1,2,6,7-3H]aldosterone were obtained from Amersham International (Les Ulis, France). BSA (fraction V) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Indo-1 acetoxymethyl ester (indo-1/AM) was purchased from Molecular Probes, Inc. (Eugene, OR). H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) was obtained from ICN Biomedicals, Inc. (Orsay, France). U-73122 [1-(6-[(17ß-3-methoxyestra-1,3,5-(10)-trien-17-yl)amino]hexyl)1H-pyrrole-2,5-dione] was obtained from Biomol (Plymouth Meeting, PA).

cAMP measurement
Adrenal slices were preincubated for 10 min at 24 C in Ringer’s solution (15 mM HEPES buffer, 112 mM NaCl, 15 mM NaHCO3, 2 mM CaCl2, and 2 mM KCl) supplemented with 2 g/liter glucose and 0.3 g/liter BSA. To inhibit phosphodiesterase activity, 3-isobutyl-1-methylxanthine (10-4 M) was added to the Ringer’s solution. The tissue slices were incubated for 20 min in the absence or presence of graded concentrations of TTN (10-9–10-6 M) as previously described (30). The reaction was stopped by adding 150 µl ice-cold 5% perchloric acid. Tissues were then homogenized and centrifuged (13,000 x g, 2 min, 4 C). The supernatant was neutralized with 1 M KHCO3, diluted with acetate buffer (0.05 M), and stored at -20 C until assay. The amount of cAMP contained in each sample was determined using a commercial kit, as previously described (31). The detection limit of the assay was 12 fmol/tube. The pellet was used for protein quantification by the Lowry method. Statistical significance was assessed by the nonparametric Mann-Whitney test.

Measurement of inositol phosphates (IPs)
Adrenal slices were incubated at 21 C for 18 h with myo-[3H]inositol (10 µCi/ml) in L15 cultured medium adjusted to R. ridibunda osmolality (L15/water = 1:0.4), supplemented with 200 mg/liter glucose, 63 mg/liter CaCl2, 1% kanamycin, and 1% antimycotic-antibiotic solutions (f-L15; pH 7.4). The tissue slices were washed six times with f-L15 medium supplemented with 1 mM inositol. After a 10-min preincubation with LiCl (10 mM), adrenal slices were incubated with TTN (10-6 M) or ET-1 (10-8 M) for 30 min. The reaction was stopped by removing the medium and adding 500 µl ice-cold 10% trichloroacetic acid. Tissues were then homogenized and centrifuged (13,000 x g, 10 min, 4 C). The supernatant that contained IPs was washed three times with water-saturated diethyl ether, neutralized with 1 M NaHCO3, and collected for analysis by anion exchange chromatography. Free inositol and total tritiated IPs were separated by anion exchange chromatography (AG1-X8 resin; 100–200 mesh; formate form; Bio-Rad Laboratories, Inc., Richmond, CA) using distilled water and a solution of 0.8 M ammonium formate in formic acid (0.1 M), respectively. The 3H radioactivity contained in each fraction was quantified by scintillation counting. Statistical significance was assessed by the nonparametric Mann-Whitney test.

Dispersion of adrenal cells
Adrenal glands were dissected, sliced, and preincubated in 5 ml f-L15. The adrenal cells were then enzymatically dispersed at 24 C for 45 min in f-L15 medium containing collagenase (2 mg/ml) and protease (2 mg/ml). After digestion, the tissue was disaggregated by gentle aspiration through a Pasteur pipette with a flame-polished tip. The suspension was centrifuged (50 x g, 15 min), and the cells were rinsed twice with fresh f-L15 medium and centrifuged.

Perifusion of dispersed adrenal cells
The effect of TTN on corticosteroid secretion was studied using a perifusion system technique for frog adrenal cells, as previously described (32). Dispersed cells were resuspended in 2 ml Ringer’s solution and transferred into the perifusion chamber (750,000 cells/chamber) between several beds of Bio-Gel P2 (Bio-Rad Laboratories, Inc.). The adrenal cells were continuously perifused with Ringer’s solution at a constant flow rate (200 µl/min) and temperature (24 C). The experimental procedure started after a stabilization period of 3 h. The viability of dispersed adrenal cells was assessed by the trypan blue exclusion test.

It has been previously shown that administration of repeated pulses of TTN to perifused adrenal tissue causes a significant attenuation of the secretory response (28). Given this desensitization phenomenon, a single pulse of TTN (10-6 M; 40 min) was administered to each perifusion column. As the responses of frog adrenocortical cells show seasonal variations in response to corticotropic factors, experiments in the absence (control) or presence of selective blockers were performed on the same day.

Corticosteroid RIAs
Corticosterone and aldosterone concentrations were determined by RIA, without prior extraction, in 200- to 300-µl samples of the same perifusion fractions, as previously described (33, 34). Direct measurement of corticosterone and aldosterone has been validated by RIA quantification of corticosteroids after HPLC analysis of the effluent perifusate (35). The working ranges of the assays were 20–5000 pg for corticosterone and 5–2000 pg for aldosterone. For both assays, the intra- and interassay coefficients of variation were less than 4% and less than 10%, respectively. None of the test substances showed any interference in the corticosterone and aldosterone RIAs.

Each perifusion pattern was established as the mean profile of corticosteroid release (±SEM) calculated over at least three independent experiments. The levels of corticosterone and aldosterone secretion were expressed as percentages of the basal values calculated as the mean of eight samples (40 min) collected at the beginning of the perifusion experiment. To compare the increase in steroid production induced by TTN in control conditions or during administration of specific blockers, the areas under the curves (AUCs) were calculated using the trapezoidal rule, as previously described (36). Statistical significance was assessed by the nonparametric Mann-Whitney test for comparison of AUC values.

Cell culture
Primary cultures of frog adrenal cells were prepared as previously described (37). Briefly, dispersed adrenal cells were suspended in 2 ml f-L15 medium and plated onto glass coverslips in petri dishes at a density of 500,000 cells/ml. The culture medium was replaced by fresh f-L15 every day.

Measurement of cytoplasmic Ca2+concentration
The effects of test substances on intracellular calcium concentration ([Ca2+]i) was studied on single adrenocortical cells by microfluorimetry, as previously described (37). Briefly, 3- to 5-day-old cultured cells were incubated at 24 C with 5 mM indo-1/AM in f-L15 medium for 40 min in the dark. The cells were washed with fresh medium and fitted to the stage of a Nikon Diaphot inverted microscope (Nikon, Melville, NY). The microscope was employed in the epifluorescence mode with an oil immersion objective (x100 CF fluor series). A pressure ejection system was used to deliver test substances in the vicinity of individual cells. The tip of the ejection glass micropipette was placed next to the cell at a distance of about 100 µm. The fluorescence emission of indo-1, induced by excitation at 355 nm (xenon lamp), was recorded at two wavelengths (405 nm corresponding to the calcium-complexed form and 480 nm corresponding to the free form) by separate photometers (P1, Nikon). The 405 nm/480 nm ratio (R) was determined using an analogical divider (constructed by Dr. B. Dufy, Bordeaux, France). All three signals (405 nm, 480 nm and R) were continuously recorded with a three-channel voltage recorder (BD 100/101, Kipp & Zonen, Delft, The Netherlands). [Ca2+]i was calculated according to the equation of Grynkiewicz et al. (38): [Ca2+]i = Kd ß[(R - Rmin)/(Rmax - R)], where Kd is the dissociation constant for indo-1 (250 nM) (38), Rmin is the fluorescence ratio obtained after incubation of cells with f-L15 containing 10 mM EGTA and 10 mM ionomycin for 3 h, Rmax is the fluorescence ratio obtained after incubation of cells with f-L15 containing 10 mM CaCl2 and 10 mM ionomycin for 3 h, and ß is the ratio between the minimal and maximal [Ca2+]i at 480 nm. The average values of Rmin, Rmax, and ß in frog adrenocortical cells were 0.143 ± 0.006 (n = 30), 1.197 ± 0.030 (n = 20), and 1.64 (n = 25), respectively. Statistical significance was assessed by the nonparametric Mann-Whitney test for comparison of values for the increase in [Ca2+]i induced by TTN in the absence or presence of test substances.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of TTN on cAMP and IP formation
Incubation of frog adrenal slices with graded concentrations of TTN (10-9 to 10-6 M) induced a dose-related increase in cAMP formation (Fig. 1AGo). In contrast, TTN (10-6 M; 30 min) did not modify the formation of total IPs (Fig. 1BGo). As a control, ET-1 (10-8 M) produced a significant increase (P < 0.01) in IP formation by frog adrenal slices.



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  		Figure 1. A, Effects of increasing concentrations of TTN on cAMP formation by frog adrenal explants. B, Effects of TTN (10-6 M) and ET-1 (10-8 M) on the formation of total IPs by frog adrenal explants. Results are expressed as percentages of the control levels. The values are the mean (±SEM) of five independent experiments. The mean cAMP concentration in basal conditions was 15.3 ± 1.1 pmol/mg protein. The mean IP level in basal conditions was 636 ± 12.4 cpm/mg protein. **, P < 0.01 vs. control.

 
Effects of G protein toxins, and protein kinase A (PKA) and phospholipase C inhibitors on TTN-induced corticosteroid secretion
In control conditions, administration of TTN provoked a significant increase in corticosteroid secretion (Fig. 2Go, A and B). Pretreatment of adrenal cells with cholera toxin (1 µg/ml; 18 h) provoked a significant decrease in TTN-evoked corticosterone (P < 0.01; Fig. 2Go, A and C) and aldosterone (P < 0.01; Fig. 2Go, B and D) secretion. Conversely, pretreatment of adrenal cells with pertussis toxin (1 µg/ml; 18 h) did not affect the stimulatory action of TTN on corticosterone (Fig. 2Go, A and E) and aldosterone (Fig. 2Go, B and F) secretion. Prolonged infusion of the protein kinase A inhibitor H-89 (10-5 M; 200 min) to perifused adrenal cells did not affect the basal secretion of corticosteroids, but significantly inhibited (P < 0.01) the stimulatory effect of TTN on corticosterone (Fig. 3Go, A and C) and aldosterone (Fig. 3Go, B and D) secretion. Administration of the phospholipase C inhibitor U-73122 (10-6 M; 200 min) did not modify the stimulatory effect of TTN on corticosterone (Fig. 4Go, A and C) and aldosterone (Fig. 4Go, B and D) secretion. As a control, U-73122 inhibits ET-1-induced stimulation of corticosteroid output from perifused frog adrenocortical tissue (39).



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  		Figure 2. Effects of G protein toxins on TTN-evoked corticosteroid secretion by perifused frog adrenal cells. A and B, Control experiments showing the effect of TTN (10-6 M; 40 min) on corticosterone (A) and aldosterone (B) secretion. C and D, Effects of TTN on corticosterone (C) and aldosterone (D) secretion after preincubation with cholera toxin (CTX; 1 µg/ml; 18 h). E and F, Effects of TTN on corticosterone (E) and aldosterone (F) secretion after preincubation with pertussis toxin (PTX; 1 µg/ml; 18 h). The profiles represent the mean (±SEM) secretion pattern of three independent perifusion experiments. Each point is the mean corticosteroid production (expressed as a percentage of spontaneous steroid output) of two consecutive fractions collected during 5 min. The spontaneous level of steroid release (100% basal level) was calculated as the mean of the first eight samples collected ({circ}). To compare the net increase in steroid production induced by TTN in control conditions and after pretreatment of the cells with CTX or PTX, the AUCs were calculated using the trapezoidal rule. The open arrows indicate the limits of the peaks that were used to calculate the AUCs. The lower panels show the AUCs in control conditions (C) and after pretreatment with CTX or PTX. The mean basal levels of corticosterone and aldosterone secretion in these experiments were 28.1 ± 2.6 and 7.4 ± 0.7 pg/min·105 cells, respectively. **, P < 0.01.

 


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  		Figure 3. Effect of TTN alone or during prolonged infusion of the PKA inhibitor H-89 on corticosteroid secretion by perifused frog adrenal cells. A and B, Control experiments showing the effects of TTN (10-6 M; 40 min) on corticosterone (A) and aldosterone (B) secretion. C and D, Effects of TTN during infusion of H-89 (10-5 M; 200 min) on corticosterone (C) and aldosterone (D) secretion. The pulse of TTN was administered 90 min after the onset of H-89 infusion. The profiles represent the mean (±SEM) secretion pattern of four independent perifusion experiments. The lower panels show the AUCs in control conditions (C) and during infusion of H-89 (H-89). The mean basal levels of corticosterone and aldosterone secretion in these experiments were 12.4 ± 1.7 and 10.2 ± 2.2 pg/min·105 cells, respectively. See Fig. 2Go for other designations. **, P < 0.01.

 


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  		Figure 4. Effect of TTN alone or during prolonged infusion of the phospholipase C inhibitor U-73122 on corticosteroid secretion by perifused frog adrenal cells. A and B, Control experiments showing the effect of TTN (10-6 M; 40 min) on corticosterone (A) and aldosterone (B) secretion. C and D, Effect of TTN during infusion of U-73122 (10-6 M; 200 min) on corticosterone (C) and aldosterone (D) secretion. The pulse of TTN was administered 90 min after the onset of U-73122 infusion. The profiles represent the mean (±SEM) secretion pattern of four independent perifusion experiments. The lower panels show the AUCs in control conditions (C) and during infusion of U-73122 (U-73). The mean basal levels of corticosterone and aldosterone secretion in these experiments were 13.8 ± 1.4 and 4.7 ± 0.8 pg/min·105 cells, respectively. See Fig. 2Go for other designations.

 
Effect of TTN on [Ca2+]i in cultured adrenocortical cells
Under resting conditions, the mean [Ca2+]i in cultured adrenocortical cells was 25 ± 2.1 nM (n = 51). Ejection of TTN (10-6 M) in the vicinity of adrenocortical cells elicited a robust increase in [Ca2+]i in 67% of the cells studied (n = 51). In all responding cells (n = 34), TTN caused a sustained and significant (P < 0.0001) increase in [Ca2+]i which peaked at 231.5 ± 17.9 nM within 30.2 ± 3.9 sec and gradually declined during the next 2 min (Fig. 5AGo). Ejection of f-L15 medium alone did not modify [Ca2+]i (Fig. 5AGo). When repeated pulses of TTN (10-6 M) were administered at 3-min intervals, a gradual attenuation of the response was observed (Fig. 5BGo). In contrast, ejection of ODN (10-6 M) in the vicinity of adrenocortical cells had no effect on [Ca2+]i (Fig. 5CGo).



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  		Figure 5. Effects of TTN and ODN on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of TTN (10-6 M; 5 sec). B, Typical profile illustrating the effect of repeated pulses of TTN (10-6 M; 5 sec). C, ODN (10-6 M; 5 sec) had no effect on [Ca2+]i. Ejection of f-L15 culture medium alone (5 sec) was used as a control. Arrows indicate the onset of each pulse of TTN, ODN, or f-L15.

 
Source of calcium involved in the TTN-induced [Ca2+]i rise
Addition of 6 mM EGTA to the f-L15 solution reduced the concentration of free calcium in the culture medium from 1.3 mM to 8 nM. After a 15-min incubation with EGTA, the mean [Ca2+]i in adrenocortical cells was reduced from 25 ± 2.1 to 12 ± 2 nM (n = 15), and the stimulatory effect of TTN on [Ca2+]i was abolished (Fig. 6Go). A brief ejection of the calcium ATPase inhibitor thapsigargin (10-5 M; 5 sec), 3 min after the TTN pulse, induced a substantial increase in [Ca2+]i (P < 0.01; Fig. 6Go). Preincubation of the cells with the T-type calcium channel blocker mibefradil (10-6 M; 10 min) significantly reduced the rise in [Ca2+]i evoked by TTN (P < 0.05; Fig. 7Go). In contrast, preincubation of the cells with the L-type calcium channel blocker nifedipine (10-6 M; 10 min) did not modify the stimulatory effect of TTN on [Ca2+]i (Fig. 8Go). Similarly, the N-type calcium channel blocker {omega}-conotoxin GVIA (10-6 M; 60 min) did not affect the [Ca2+]i response evoked by TTN (Fig. 9Go).



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  		Figure 6. Effect of TTN (10-6 M; 5 sec) and thapsigargin (10-5 M; 5 sec) in the presence of EGTA (6 mM) on [Ca2+]i in cultured frog adrenocortical cells. The cells were incubated with EGTA 15 min before administration of the TTN pulse. Thapsigargin was used to test the state of depletion of intracellular Ca2+ stores. Arrows indicate the onset of the pulses of TTN or thapsigargin. The lower panel shows the mean values of [Ca2+]i in normal f-L15 medium (control; C) and f-L15 medium supplemented with 6 mM EGTA (EGTA), and after application of TTN or thapsigargin (thapsi) pulses in EGTA-supplemented medium. **, P < 0.01.

 


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  		Figure 7. Effect of TTN (10-6 M; 5 sec) in the absence or presence of the T-type calcium channel blocker mibefradil on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of TTN before incubation of the cells with mibefradil. B, Effect of TTN in the presence of mibefradil (10-6 M). Mibefradil was added to the incubation medium 10 min before application of the pulse of TTN. The inset shows the mean increase in [Ca2+]i ({Delta}[Ca2+]i) induced by TTN in normal f-L15 medium (control; C) and in the presence of mibefradil (Mib). *, P < 0.05.

 


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  		Figure 8. Effect of TTN (10-6 M; 5 sec) in the absence or presence of the L-type calcium channel blocker nifedipine on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of TTN before incubation of the cells with nifedipine. B, Effect of TTN in the presence of nifedipine (10-6 M). Nifedipine was added to the incubation medium 10 min before application of the pulse of TTN. The inset shows the mean increase in [Ca2+]i ({Delta}[Ca2+]i) induced by TTN in normal f-L15 medium (control; C) and in the presence of nifedipine (Nif).

 


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  		Figure 9. Effect of TTN (10-6 M; 5 sec) in the absence or presence of the N-type calcium channel blocker {omega}-conotoxin GVIA on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of TTN before incubation of the cells with {omega}-conotoxin. B, Effect of TTN in the presence of {omega}-conotoxin (10-6 M). {omega}-Conotoxin was added to the incubation medium 60 min before application of the pulse of TTN. The inset shows the mean increase in [Ca2+]i ({Delta}[Ca2+]i) induced by TTN in normal f-L15 medium (control; C) and in the presence of {omega}-conotoxin ({omega}-con).

 
Preincubation of the cells with the PKA inhibitor H-89 (10-5 M; 90 min) significantly reduced the effect of TTN on [Ca2+]i (Fig. 10Go, A and B). After a washout period of 30 min in the absence of H-89, the stimulatory effect of TTN was totally recovered (Fig. 10CGo).



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  		Figure 10. Effect of TTN (10-6 M; 5 sec) in the absence or presence of the PKA inhibitor H-89 on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of TTN before incubation of the cells with H-89. B, Effect of TTN in the presence of H-89 (10-5 M). H-89 was added to the incubation medium 90 min before application of the pulse of TTN. C, Effect of TTN 30 min after withdrawal of H-89. The inset shows the mean increase in [Ca2+]i ({Delta}[Ca2+]i) induced by TTN in normal f-L15 medium (control; C), in the presence of H-89 (H-89) and after a washout period of 30 min (W). ***, P < 0.001.

 
Requirement of Ca2+ for TTN-induced steroid secretion
Prolonged infusion of mibefradil (10-6 M; 200 min) to perifused adrenal cells did not modify the basal secretion of corticosteroids, but significantly inhibited the stimulatory effect of TTN on corticosterone (P < 0.01; Fig. 11Go, A and C) and aldosterone (P < 0.01; Fig. 11Go, B and D) secretion. Conversely, in the same conditions, nifedipine (10-6 M; 200 min) did not affect the TTN-evoked stimulation of corticosterone secretion (Fig. 12Go).



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  		Figure 11. Effect of TTN alone or during prolonged infusion of the T-type calcium channel blocker mibefradil on corticosteroid secretion by perifused frog adrenal cells. A and B, Control experiments showing the effect of TTN (10-6 M; 40 min) on corticosterone (A) and aldosterone (B) secretion. C and D, Effect of TTN during infusion of mibefradil (10-6 M; 200 min) on corticosterone (C) and aldosterone (D) secretion. The pulse of TTN was administered 90 min after the onset of mibefradil infusion. The profiles represent the mean (±SEM) secretion pattern of three independent perifusion experiments. The lower panels show the AUCs in control conditions (C) and during infusion of mibefradil (Mib). The mean basal levels of corticosterone and aldosterone secretion in these experiments were 24.8 ± 3.5 and 6.3 ± 1.6 pg/min·105 cells, respectively. See Fig. 2Go for other designations. **, P < 0.01.

 


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  		Figure 12. Effect of TTN alone or during prolonged infusion of the L-type calcium channel blocker nifedipine on corticosterone secretion by perifused frog adrenal cells. A, Control experiments showing the effect of TTN (10-6 M; 40 min) on corticosterone secretion. B, Effect of TTN during infusion of nifedipine (10-6 M; 200 min) on corticosterone secretion. The pulse of TTN was administered 90 min after the onset of nifedipine infusion. The profiles represent the mean (±SEM) secretion pattern of three independent perifusion experiments. The lower panel shows the AUCs in control conditions (C) and during infusion of nifedipine (Nif). The mean basal level of corticosterone secretion in these experiments was 11.9 ± 0.3 pg/min·105 cells. See Fig. 2Go for other designations.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously reported the occurrence of endozepine-like immunoreactivity in frog adrenochromaffin cells (27), and we have demonstrated that TTN is a potent stimulator of corticosterone and aldosterone secretion (28), suggesting that endozepines may act as paracrine factors participating in the local regulation of adrenal steroidogenic cells by chromaffin cells (40, 41, 42). In the present study, we have investigated the transduction pathways involved in the mechanism of action of TTN in frog adrenocortical cells.

Involvement of adenylyl cyclase and phospholipase C in the corticotropic action of TTN
We have previously shown that the stimulatory effect of TTN on corticosteroid secretion by the frog adrenal gland is not mediated through activation of central- or peripheral-type benzodiazepine receptors (28). The present study now reveals that TTN stimulates cAMP formation and has no effect on polyphosphoinositide metabolism. Our data also showed that preincubation of frog adrenal cells with cholera toxin almost completely abolished the effect of TTN on corticosterone and aldosterone secretion, whereas pretreatment with pertussis toxin had no effect. These observations indicate that the stimulatory effect of TTN on adrenocortical cells can be ascribed to activation of a membrane receptor positively coupled to adenylyl cyclase through a cholera toxin-sensitive G protein. It has been recently reported that in rat glial cells, ODN, another DBI-derived peptide, stimulates a membrane receptor positively coupled to phospholipase C (43). Taken together, these data indicate that endozepines, which were originally characterized as endogenous ligands of benzodiazepine receptors (1), can also modulate the activity of metabotropic membrane receptors coupled to either adenylyl cyclase or phospholipase C.

We next investigated the possible involvement of adenylyl cyclase and phospholipase C in the corticotropic action of TTN. Pretreatment of adrenal cells with the PKA inhibitor H-89 markedly reduced the stimulatory effect of TTN on corticosterone and aldosterone secretion. In contrast, the phospholipase C inhibitor U-73122 did not impair the TTN-evoked stimulation of steroid output. These data show that in frog adrenocortical cells, the stimulatory effect of TTN on corticosteroid secretion can be accounted for by activation of the adenylyl cyclase/PKA pathway.

Involvement of cytosolic Ca2+ in the corticotropic action of TTN
In contrast to mammals, the frog adrenal gland does not exhibit any zonation, but is composed of steroidogenic cells intermingled with chromaffin cells (30, 32); thus, frog adrenal cells in culture are composed of a mixed population of adrenocortical and chromaffin cells. However, the two cell types can be easily identified under the microscope on the basis of their morphological characteristics (30, 37). In resting conditions, the average [Ca2+]i in frog adrenocortical cells was relatively low (25 ± 2.1 nM; n = 51), and the cells did not display oscillatory transients as observed in bovine (44) and human adrenocortical cells (45). In frog adrenocortical cells, TTN caused a biphasic increase in [Ca2+]i, i.e. a transient stimulation followed by a prolonged plateau phase. In contrast, the endozepine ODN did not modify [Ca2+]i. Consistent with this observation, we have previously found that ODN, contrary to TTN, does not stimulate steroid secretion in the frog adrenal gland (28). Administration of repeated pulses of TTN resulted in a sequential stimulation of [Ca2+]i with gradual attenuation of the response. This desensitization process is reminiscent of the tachyphylaxis of the secretory response observed during the administration of repeated pulses of TTN to frog adrenal slices (28). A similar attenuation of the Ca2+ response was observed during iterative administration of other corticotropic neuropeptides, such as pituitary adenylate cyclase-activating polypeptide (30) and ranakinin (46).

In mammalian adrenocortical cells, calcium influx through voltage-dependent Ca2+ channels plays a crucial role in the response to corticotropic factors (47, 48, 49). The present data show that suppression of free Ca2+ in the incubation medium with EGTA abrogated the increase in [Ca2+]i evoked by TTN. The blockage of the Ca2+ response cannot be ascribed to depletion of intracellular Ca2+ stores, because the Ca2+-adenosine triphosphatase inhibitor thapsigargin could still produce a massive increase in [Ca2+]i in the presence of EGTA. Moreover, suppression of calcium in the perifusion medium significantly attenuated the stimulatory effect of TTN on corticosteroid secretion (data not shown), indicating that the corticotropic action of TTN can be accounted for at least in part by calcium influx through the plasma membrane.

Characterization of the calcium channel mediating the stimulatory effect of TTN
Preincubation of adrenal cells with 10-6 M mibefradil, a benzimidazolyl-substituted tetraline derivative exhibiting relative selectivity for T-type calcium channels (47, 50), strongly attenuated the stimulatory effect of TTN on [Ca2+]i. In contrast, at the same concentration, the L-type calcium channel blocker nifedipine and the N-type calcium channel blocker {omega}-conotoxin did not affect the Ca2+ response of adrenocortical cells to TTN. The fact that the transient [Ca2+]i increase and the sustained plateau phase were both strongly reduced by mibefradil suggests that the effect of TTN on Ca2+ influx is entirely mediated through T-type calcium channels. Mibefradil also markedly attenuated the stimulatory effect of TTN on corticosteroid secretion, indicating that Ca2+ influx through T-type calcium channels is required for the secretory response of frog adrenocortical cells to TTN. Consistent with these observations, several reports have demonstrated the involvement of T-type calcium channels in the mechanism of action of various corticotropic factors, including angiotensin II, potassium, and serotonin (36, 47, 48).

As TTN caused both stimulation of cAMP formation and an increase in [Ca2+]i, the next step of our study was to determine the sequence of events associated with the action of TTN on adrenocortical cells. Preincubation of adrenal cells with H-89 markedly attenuated the stimulatory effect of TTN on [Ca2+]i, indicating that a cAMP-dependent protein kinase is responsible for the activation of T-type calcium channels. The observation that the calcium response to TTN was delayed by 30 sec is consistent with an indirect effect of TTN on the [Ca2+]i rise, i.e. secondary to the activation of the cAMP-PKA pathway. However, we cannot exclude that the Ca2+ influx evoked by TTN could result from depolarization of frog adrenocortical cells as previously reported for ACTH-induced stimulation of bovine fasiculata cells (51).

A proposed model illustrating the mechanism of action of TTN on adrenocortical cells is shown in Fig. 13Go. TTN, contained within chromaffin cells, stimulates a novel endozepine receptor positively coupled to adenylyl cyclase through a cholera toxin-sensitive G protein. The activation of a cAMP-dependent protein kinase subsequently increases calcium entry through T-type calcium channels. Both the activation of the adenylyl cyclase/PKA pathway and the calcium influx appear to be involved in the stimulatory effect of TTN on corticosteroid secretion.



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  		Figure 13. Schematic representation summarizing the mechanism of action of TTN in the frog adrenal gland. Activation of the TTN receptor increases adenylyl cyclase activity via a cholera toxin-sensitive G protein-coupled receptor. The resulting activation of a PKA causes stimulation of calcium influx through T-type calcium channels. Calcium entry is required for the stimulatory effect of TTN on corticosterone and aldosterone secretion.

 


   Acknowledgments
 
We thank Dr. A. Fournier, INRS Santé (Montréal, Québec, Canada), for the kind gift of synthetic endothelin-1. The expert technical assistance of Mrs. H. Lemonnier is gratefully acknowledged.


   Footnotes
 
1 This work was supported by grants from INSERM (U-413) and the Conseil Régional de Haute-Normandie. Back

2 Invited Professor from the University of Benin, Faculty of Sciences, Lome, Togo. Back

Received March 8, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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