Activation of EndothelinA Receptors in Frog Adrenocortical Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium
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内分泌学杂志 2005年第1期
European Institute for Peptide Research (Institut Fédratif de Recherches Multidisciplinaires sur les Peptides 23) (C.D., I.R.J., M.G., L.G., H.V.), Laboratory of Cellular and Molecular Neuroendocrinology (Institut National de la Santé et de la Recherche Médicale U 413), University of Rouen, 76821 Mont-Saint-Aignan, France; and Institut National de la Recherche Scientifique-Santé/Institut Armand Frappier, Université du Québec (A.F.), Pointe-Claire, Québec, Canada H9R 1G6
Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, European Institute for Peptide Research (Institut Fédratif de Recherches Multidisciplinaires sur les Peptides 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U 413, UA Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry@univ-rouen.fr.
Abstract
We have previously shown that endothelin (ET)-1 stimulates corticosterone and aldosterone secretion by the frog adrenal gland through activation of ETA receptors positively coupled to both the adenylyl cyclase and phospholipase C (PLC) pathways. The purpose of the present study was to investigate the involvement of calcium in ET-1-induced stimulation of corticosteroid secretion. Cytoautoradiographic labeling using [125I]ET-1 as a tracer revealed the presence of ET-1 binding sites on adrenocortical cells. Administration of graded concentrations of ET-1 in the vicinity of adrenocortical cells provoked a dose-dependent increase in cytosolic calcium concentrations ([Ca2+]i). ET-1 induced a biphasic response consisting of an immediate and transient peak of [Ca2+]i followed by a plateau phase. Preincubation of the cells with the calcium-ATPase inhibitor thapsigargin or the PLC inhibitor U-73122 reduced the amplitude of the transient phase. Administration of the calcium chelator EGTA or the protein kinase A inhibitor H-89 attenuated the plateau phase. The [Ca2+]i response to ET-1 was markedly reduced during concomitant administration of U-73122 and H-89. Preincubation of the cells with the L-type calcium channel blocker nifedipine attenuated the plateau phase. Corticosteroid secretion from perifused frog adrenal slices was almost completely suppressed by thapsigargin and reduced by nifedipine. Taken together, these data indicate that activation of ETA receptors in frog adrenocortical cells provokes immediate stimulation of PLC, which causes an early mobilization of calcium from intracellular stores, and activates adenylyl cyclase, which results in delayed calcium influx through L-type calcium channels. The resulting increase in [Ca2+]i plays a pivotal role in ET-1-induced corticosteroid secretion.
Introduction
THE ENDOTHELIN (ET) FAMILY comprises three isoforms of vasoactive peptides (ET-1, ET-2, and ET-3), which all possess 21 amino acids including four cysteine residues that form two intracellular disulfide bonds (1, 2). The primary structure of ETs has been strongly preserved during evolution. Notably, the sequence of ET-1 is identical in frog and human (3). The effects of ETs on smooth muscle cells are mediated through two types of G protein-coupled receptors that exhibit differential affinities for the isopeptides: the ETA receptor possesses higher affinity for ET-1 and ET-2 than for ET-3, whereas the ETB receptor has similar affinity for all three ET isoforms (4).
Besides the vascular bed, ET receptors are expressed in various endocrine and neuroendocrine tissues, and ETs modulate the secretion of a number of hormones (see Ref. 5 for review). In particular, ETs are potent activators of corticosteroid secretion in all vertebrate species investigated so far including human, bovine, rabbit, rat, and frog (6). However, the type of receptor mediating the effect of ETs on adrenocortical cells markedly differs from one species to the other. For instance, in the human adrenal gland, both ETA and ETB receptors are involved in the stimulatory effect of ET-1 on cortisol secretion (7). In rat, the stimulatory effect of ET-1 on fasciculata cells is mediated via ETB receptors (8), whereas the action of ET-1 on mineralocorticoid secretion has been ascribed to activation of either the ETB receptor only (8) or both the ETA and ETB receptors (9). In contrast, in frog, the effect of ET-1 on corticosteroid secretion is exclusively mediated through ETA receptors (10). The amphibian adrenal gland, which is composed of a single population of adrenocortical cells secreting both corticosterone and aldosterone, is a very suitable model in which to investigate the signaling mechanisms associated with activation of ETA receptors. A particular advantage of a pure population of endocrine cells, such as frog adrenocortical cells, in studying the transduction pathways downstream ETA receptors is that the effect of ET-1 on second messenger systems can be readily correlated with the final response of the cell by measuring corticosteroid secretion.
We previously demonstrated that, in frog adrenocortical cells, the stimulatory effect of ET-1 on corticosteroid secretion is mediated through activation of adenylyl cyclase (AC) and phospholipase C (PLC) (11). We have also shown that corticotropic agents stimulating AC can provoke calcium (Ca2+) influx through T-type Ca2+ channels (12, 13). Thus, in the present study, we investigated the effect of ETA receptor activation on calcium mobilization in frog adrenocortical cells in primary culture. The involvement of calcium in ET-1-induced corticosterone and aldosterone secretion has also been studied on perifused frog adrenal slices.
Materials and Methods
Reagents and test substances
Synthetic ET-1 (frog/human sequence) and ranakinin (frog substance P ortholog) were synthetized by the solid-phase methodology as previously described (14, 15). BQ-485 (perhydroazepin-L-yl-L-leucyl-D-tryptophanyl-D-tryptophan) was a generous gift from Banyu Pharmaceuticals (Tsukuba, Japan). Mibefradil (dihydrochloride) was kindly provided by Hoffmann-La Roche (Basel, Switzerland). The antiserum against 3?-hydroxysteroid dehydrogenase (3?-HSD) was generously provided by V. Luu-The (Laval University, Québec, Canada). Fluorescein isothiocyanate-conjugated goat antirabbit -globulins were purchased from Nordic Immunological Laboratories (Tilburg, The Netherlands). Leibovitz culture medium (L15), HEPES, thapsigargin, EGTA, collagenase (type IA), protease (from Bacillus polymyxa; type IX), nifedipine, -conotoxin GVIA, ionomycin, and BSA were obtained from Sigma (St. Louis, MO). Fetal bovine serum, the kanamycin solution (10,000 U/ml), and the antibiotic-antimycotic solution (penicillin G sodium, 10,000 U/ml; streptamycin sulfate, 10,000 U/ml) were from Life Technologies, Inc. (Grand Island, NY). 3-[125I]iodotyrosyl-ET-1 (2000 Ci/mmol), [1, 2, 6, 7-3H]corticosterone, and [1, 2, 6, 7-3H]aldosterone were from Amersham International (Les Ulis, France). Indo-1-pentacetoxymethylester was purchased from Molecular Probes (Leiden, The Netherlands). H-89, N-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinoline-sulfonamide, was from ICN Pharmaceuticals (Orsay, France). U-73122 [1-(6-[(17?-3-methoxyestra-1,3,5-(10)-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione] was from Biomol Research Laboratories (Plymouth Meeting, PA).
Animals
Adult male frogs (Rana esculenta; body weight 30–40 g) originating from Albania were obtained from a commercial supplier (Couétard, Saint-Hilaire de Riez, France). The animals were maintained in glass tanks supplied with a trickle of tap water, in a temperature-controlled room (8 ± 1 C) on a 12-h dark, 12-h light regimen (lights on from 0600 to 1800 h) for at least 1 wk before use. Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.
Cell culture
Frogs were killed by decapitation, and the adrenal glands were dissected free of renal parenchyma. The glands were washed three times with L15 medium adjusted to R. esculenta osmolality (L15/water = 1:0.4) supplemented with 0.4 mM CaCl2, 15 mM HEPES, 0.2 mM glucose, and 1% each of the kanamycin and antibiotic-antimycotic solutions (f-L15; pH 7.4). Adrenal cells were then enzymatically dispersed as previously described (16). Briefly, eight pairs of adrenal glands were subjected to enzymatic dissociation at 24 C for 45 min in an f-L15 solution 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. Dispersed cells were centrifuged (50 x g; 5 min) and rinsed three times with f-L15. The cells were suspended in 2 ml f-L15 medium supplemented with 10% fetal bovine serum and plated onto glass coverslips in petri dishes. The cells were kept at 22 C in an incubator with a humidified atmosphere. The culture medium was renewed 24 h after plating and subsequently every 48 h.
Immunofluorescence procedure
Immunohistochemical identification of adrenocortical cells (vs. chromaffin cells) was carried out on 3-d-old cells using the indirect immunofluorescence method, as previously described (16). The culture medium was aspirated and the cells were rinsed three times with 0.1 M phosphate buffer (PB; pH 7.4). The cells were fixed for 2 h at room temperature with a solution of 4% paraformaldehyde in PB and rinsed again three times with PB. The cells were then incubated 2 h at room temperature with the antiserum against 3?-HSD (1:100). The antiserum was diluted in PB with 1% BSA and 0.3% Triton X-100. The cells were rinsed with PB and incubated for 1 h at room temperature with fluorescein isothiocyanate-conjugated goat anti-rabbit -globulins (1:100). Finally, the cells were rinsed in PB, mounted with PB-glycerol (1:1), and coverslipped. The preparations were examined on a Orthoplan microscope (Leica, Heidelberg, Germany).
Cytoautoradiographic studies
Cells cultured for 3 d and plated at a low density (20,000 cells/cm2) were washed with 50 mM Tris buffer (pH 7.4) containing 0.2% BSA, 5 mM MgCl2, and 0.5 μg bacitracin per milliliter. The cells were incubated with 3-[125I]iodotyrosyl-ET-1 (100 pM) for 2 h at 24 C in Tris buffer supplemented with 0.2% BSA. Nonspecific binding was determined by adding 10–6 M ET-1. The coverslips were rinsed, glued to glass slides, and exposed to paraformaldehyde vapors (60 C for 24 h). The coverslips were then dipped into NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY) at 40 C. After 6 d of exposure, the cells were counterstained with Toluidine blue and examined under a Leitz Orthoplan microscope.
Calcium measurement
The effect of test substances on cytosolic calcium concentration ([Ca2+]i) was studied on single adrenocortical cells by a microfluorimetric technique, as previously described (16). Briefly, adrenal cells cultured for 3–5 d (70,000 cells/cm2) were incubated in darkness (24 C, 40 min) with 5 μM Indo-1-pentacetoxymethylester in f-L15 medium. The coverslips were washed with fresh medium and fitted to the stage of a Diaphot inverted microscope (Nikon, Melville, NY) equipped for epifluorescence with an oil immersion objective (x100 CF Fluor series; numerical operture 1.3). A pressure ejection system was used to deliver test substances in the vicinity of individual cells. 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 405:480 nm ratio R was determined using an analogic divider (constructed by Dr. B. Dufy, Bordeaux, France). All three signals (405 and 480 nm and R) were continuously recorded with a three-channel voltage recorder (BD 100/101, Kipp and Zonen, Delft, The Netherlands). [Ca2+]i was calculated from the formula established by Grynkiewicz et al. (17): [Ca2+]i = Kd x ? x [(R – Rmin)/(Rmax – R)], where Kd is the dissociation constant for indo-1 (250 nM), ? is the ratio of fluorescence yield from the Ca2+min/Ca2+max indicator at 480 nm, Rmin is the fluorescence ratio obtained after incubation of cells with f-L15 containing 10 mM EGTA and 10 μM ionomycin for 3 h, and Rmax is the fluorescence ratio obtained after incubation of cells with f-L15 containing 10 mM CaCl2 and 10 μM ionomycin for 3 h. The average values of ?, Rmin, and Rmax in frog adrenocortical cells were 1.77 (n = 20), 0.077 ± 0.008 (n = 15), and 1.033 ± 0.050 (n = 18), respectively.
Perifusion technique
The effect of test substances on corticosteroid secretion was studied by using a perifusion system technique, as previously described (10). For each perifusion experiment, six pairs of adrenal glands were sliced with scissors and preincubated in 5 ml Ringer’s solution (100 mM NaCl, 15 mM NaHCO3, 2 mM CaCl2, 2 mM KCl, 15 mM HEPES, 2 mg/ml glucose, and 0.3 mg/ml BSA). The Ringer’s solution was gassed with a 95% O2-5% CO2 mixture, and the pH was adjusted to 7.4. The adrenal slices were rinsed twice with Ringer’s medium and layered into a perifusion chamber between several beds of Bio-Gel P2 (Bio-Rad Laboratories, Ivry-sur-Seine, France). The adrenal slices were continuously perifused with gassed Ringer’s solution alone or with test substances, at constant flow rate (200 μl/min) and temperature (24 C). Fractions of effluent perfusate were collected every 5 min and frozen until assay. Nifedipine was dissolved in ethanol so that the final concentration of ethanol never exceeded 0.2%. Thapsigargin was dissolved in dimethylsulfoxide (DMSO) so that the final concentration of DMSO in the perifusion medium was 0.1%. Previous studies have shown that ethanol and DMSO, at concentrations of 0.2 and 0.1%, respectively, have no effect on corticosteroid secretion.
Corticosteroid RIA
Corticosterone and aldosterone concentrations were directly determined in 200- to 300-μl aliquots of each perifusion fraction without prior extraction, as previously described (18). Direct measurement of corticosterone and aldosterone has been validated by RIA quantification of corticosteroid after HPLC analysis of the effluent perfusate (19). The sensitivity thresholds of the assays were 20 pg for corticosterone and 5 pg for aldosterone. For both assays, the intra- and interassay coefficients of variation were 3 and 6% for corticosterone, and 5 and 8% for aldosterone, respectively. Each perifusion pattern was established as the mean profile of corticosteroid production (± SEM) calculated from at least three independent experiments. Corticosterone and aldosterone levels were expressed as percentages of the basal values, calculated as the mean of eight samples (40 min), taken just before the infusion of test substances.
Calculations
All data are expressed as mean ± SEM. The Mann-Whitney U test was used for comparison of the mean values between two groups. To compare the net increase in [Ca2+]i or steroid secretion induced by ET-1 in the absence or presence of various test substances, the areas under the curves (AUCs) were calculated using the trapezoidal rule (12).
Results
Identification of cultured adrenal cells
Under the light microscope, adrenocortical and chromaffin cells can be easily discriminated: the former are large, polygonal cells containing refractive lipid droplets, whereas the latter are spherical cells punctuated with secretory granules (Fig. 1A). Immunocytochemical labeling with antibodies against 3?-HSD confirmed the reliability of the morphological identification of adrenocortical cells (Fig. 1B).
FIG. 1. Micrographs of frog adrenal cells after 3 d of culture. A, Light photomicrograph showing that adrenocortical cells (arrows) are large cells containing refractive lipid droplets, whereas chromaffin cells (arrowhead) are spherical cells with punctuated granules. B, Immunocytochemical labeling of adrenocortical cells with antibodies against 3?-HSD. Bars, 10 μm.
Cellular localization of ET-1 binding sites
Cytoautoradiographic staining of cultured adrenal cells with [125I]ET-1 showed selective accumulation of silver grains on adrenocortical cells, whereas the density of grains on chromaffin cells was similar to that of the background (Fig. 2, A and B). In the presence of 10–6 M ET-1, autoradiographic labeling of adrenocortical cells was markedly reduced (Fig. 2, C and D).
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FIG. 2. Autoradiographic visualization of [125I]ET-1 binding sites on cultured frog adrenal cells. A, Photomicrograph of cultured cells stained with Toluidine blue, showing a dense accumulation of silver grains over adrenocortical cells (arrows); chromaffin cells were not labeled (arrowheads). B, Dark-field illumination micrograph of the field shown in A. C, Nonspecific binding in the presence of 10–6 M ET-1. D, Dark-field illumination micrograph of the field shown in C. Bars, 10 μm.
Effect of ET-1 on [Ca2+]i in cultured adrenocortical cells
Under resting conditions, the mean [Ca2+]i in cultured adrenocortical cells was 36.4 ± 2.1 nM (n = 328). Microejection of ET-1 (10–6 M; 5 sec) in the vicinity of adrenocortical cells provoked a marked increase in [Ca2+]i in 65% of the cells (n = 213), whereas application of vehicle had no effect (Fig. 3). In all responding cells, ET-1 evoked a biphasic response consisting of an immediate and transient peak of [Ca2+]i followed by a plateau phase (Fig. 3). Conversely, in chromaffin cells, ET-1 (10–6 M) has no effect on [Ca2+]i (n = 34), whereas the substance P analog ranakinin (10–6 M) elicited a robust increase in [Ca2+]i (Fig. 3, inset) as previously described (20). Application of graded concentrations of ET-1 (10–12 to 10–6 M) in the vicinity of adrenocortical cells produced a dose-dependent increase in [Ca2+]i with an EC50 of 1.7 x 10–9 M (Fig. 4). The minimum effective dose was 10–10 M (P < 0.05), and maximum stimulation was observed at a concentration of 10–7 M (P < 0.01). Recording of adrenocortical cells during 30 min (n = 46) revealed that ET-1 (10–6 M) induced Ca2+ oscillations in 65% of the responding cells (Fig. 5). The duration of each Ca2+ transient (81 ± 4 sec) and the mean frequency (8 x 10–3 Hz) were reproducible. In light of this oscillation phenomenon, each cell studied received a single dose of ET-1, and only one cell was recorded per petri dish. The selective ETA receptor antagonist BQ-485 (10–5 M) totally abolished the effect of ET-1 (10–6 M) on [Ca2+]i in adrenocortical cells (P < 0.001; Fig. 6).
FIG. 3. Typical profile illustrating the effect of a single application of 10–6 M ET-1 on [Ca2+]i in cultured frog adrenocortical cells. The profile is representative of 213 cells recorded, 65% of which responded to ET-1 by an increase in [Ca2+]i. Inset, ET-1 had no effect on [Ca2+]i in chromaffin cells, whereas 10–6 M ranakinin (RK), used as a control, induced a robust increase in [Ca2+]i. The arrows indicate the onset of application of culture medium alone (vehicle), ET-1, or RK.
FIG. 4. Dose-response curve showing the effect of graded concentrations of ET-1 on [Ca2+]i in cultured frog adrenocortical cells. The data have been calculated from a series of recordings similar to that presented in Fig. 3. The concentrations indicated on the x-axis are those contained in the ejection pipette. The number of cells studied at each point is indicated between parentheses. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG. 5. Effect of ET-1 (10–6 M) on [Ca2+]i oscillations in cultured frog adrenocortical cells. The profile is representative of 46 cells recorded, 65% of which responded to ET-1 by calcium transients. The arrows indicate the onset of vehicle or ET-1 application.
FIG. 6. Effect of ET-1 in the absence or presence of the selective ETA receptor antagonist BQ-485 (10–5 M) on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of 10–6 M ET-1. B, Incubation of cells with the ETA receptor antagonist BQ-485 totally suppressed the effect of ET-1 on [Ca2+]i. BQ-485 was added to the bath solution 60 min before application of the pulse of ET-1. The arrows indicate the onset of ET-1 application. The bar indicates the duration of BQ-485 administration. C, The histograms show that BQ-485 reduced by 99.3% the amplitude of the [Ca2+]i response induced by ET-1. ***, P < 0.001.
Sources of Ca2+ involved in ET-1-induced [Ca2+]i rise
Preincubation of the cells with the Ca2+ ATPase inhibitor thapsigargin (10–5 M; 5 min) induced a substantial elevation of [Ca2+]i from 28.9 ± 2.9 (n = 11) to 105.3 ± 9.5 nM (n = 13; Fig. 7, A and B). In the presence of thapsigargin, the amplitude of the early [Ca2+]i response to ET-1 was significantly reduced (P < 0.001; Fig. 7C). As a control, we have previously shown that thapsigargin does not impair serotonin-induced [Ca2+]i rise in frog adrenocortical cells (12). Concurrently, preincubation of the cells with the PLC inhibitor U-73122 (10–6 M; 60 min) did not affect basal [Ca2+]i but significantly attenuated (P < 0.01) the amplitude of the transient phase of ET-1-evoked Ca2+ increase (Fig. 8). As a control, it has been previously reported that U-73122 has no effect on serotonin-evoked [Ca2+]i increase in frog adrenocortical cells (12). Addition of 10 mM EGTA to f-L15, which reduced the concentration of free Ca2+ in the extracellular medium from 1.3 to 8 nM, provoked a significant reduction (P < 0.01) of the duration of the [Ca2+]i wave from 143 ± 17.9 to 80.1 ± 8.6 sec (Fig. 9, A and B). As a consequence, a 15-min incubation with EGTA significantly diminished (P < 0.01) the AUC of the [Ca2+]i response (Fig. 9C). Pilot experiments have shown that, in frog adrenocortical cells incubated with EGTA, thapsigargin could still provoke a robust increase in [Ca2+]i (Fig. 9B, inset), indicating that EGTA did not deplete the pool of intracellular Ca2+. In addition, we have previously shown that EGTA does not affect the response of frog adrenochromaffin cells to ranakinin (20).
FIG. 7. Effect of ET-1 in the absence or presence of the calcium ATPase inhibitor thapsigargin on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of 10–6 M ET-1. B, Effect of ET-1 in the presence of 10–5 M thapsigargin. Thapsigargin was added to the bath solution 5 min before application of the pulse of ET-1. The arrows indicate the onset of vehicle or ET-1 application. The bar indicates the duration of thapsigargin administration. C, The histograms show that thapsigargin reduced by 60.9% the amplitude of the [Ca2+]i response induced by ET-1. ***, P < 0.001.
FIG. 8. Effect of ET-1 in the absence or presence of the PLC inhibitor U-73122 on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of 10–6 M ET-1. B, Effect of ET-1 in the presence of 10–6 M U-73122. U-73122 was added to the bath solution 60 min before application of the pulse of ET-1. The arrows indicate the onset of ET-1 application. The bar indicates the duration of U-73122 administration. C, The histograms show that U-73122 reduced by 73.1% the amplitude of the [Ca2+]i response induced by ET-1. **, P < 0.01.
FIG. 9. Effect of ET-1 in the absence or presence of the Ca2+ chelator EGTA on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of 10–6 M ET-1. B, Effect of ET-1 in the presence of 10–2 M EGTA. EGTA was added to the bath solution 5 min before application of the pulse of ET-1. Inset, EGTA did not deplete the pool of intracellular Ca2+ because thapsigargin (Tg) could still provoke a massive increase in [Ca2+]i. The arrows indicate the onset of ET-1 application. The bar indicates the duration of EGTA administration. C, The histograms show that EGTA reduced by 55.1% the AUC of the [Ca2+]i response induced by ET-1. **, P < 0.01.
Administration of the protein kinase A (PKA) inhibitor H-89 (10–5 M) did not affect the amplitude of the early [Ca2+]i peak but totally suppressed the delayed phase (Fig. 10, A and B). A brief ejection of H-89 during the plateau phase provoked a rapid decrease of [Ca2+]i to basal level (Fig. 10C). During prolonged incubation with H-89, the AUC of the [Ca2+]i response was significantly reduced (P < 0.001; Fig. 10D). Concomitant incubation of adrenocortical cells with U-73122 (10–6 M) and H-89 (10–5 M) almost completely suppressed the [Ca2+]i response induced by ET-1 (P < 0.001; Fig. 11). Preincubation of adrenocortical cells with the T-type Ca2+ channel blocker mibefradil (10–6 M; 15 min; Fig. 12A) or the N-type Ca2+ channel blocker -conotoxin GVIA (10–6 M; 30 min; Fig. 12B) did not modify the [Ca2+]i response evoked by ET-1. As controls, we have previously shown that mibefradil markedly attenuates triakontatetraneuropeptide-induced [Ca2+]i rise in frog adrenocortical cells (13) and that -conotoxin GVIA reduces neurotensin-evoked [Ca2+]i rise in frog melanotrope cells (21). Conversely, the L-type channel blocker nifedipine (10–6 M; 15 min) diminished the amplitude and duration of the plateau phase without affecting the amplitude of the transient phase (Fig. 12C). Thus, nifedipine significantly reduced the AUC of the [Ca2+]i response (P < 0.01; Fig. 12C).
FIG. 10. Effect of ET-1 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 10–6 M ET-1. B and C, Effect of ET-1 in the presence of 10–5 M H-89. B, H-89 was added to the bath solution 60 min before application of the pulse of ET-1. C, H-89 was ejected during the plateau phase of the response. The arrows indicate the onset of ET-1 or H-89 application. The bar indicates the duration of H-89 administration. D, The histograms show that H-89 reduced by 75.7% the AUC of the [Ca2+]i response induced by ET-1. ***, P < 0.001.
FIG. 11. Effect of ET-1 in the absence or presence of the PLC inhibitor U-73122 and the PKA inhibitor H-89 on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of 10–6 M ET-1. B, Effect of ET-1 in the presence of 10–6 M U-73122 plus 10–5 M H-89. U-73122 and H-89 were added to the bath solution 60 min before application of the pulse of ET-1. The arrows indicate the onset of ET-1 application. The bar indicates the duration of U-73122 and H-89 administration. C, The histograms show that concomitant administration of U-73122 and H-89 reduced by 89.9% the amplitude of the [Ca2+]i response induced by ET-1. ***, P < 0.001.
FIG. 12. Effect of ET-1 in the absence or presence of selective blockers of voltage-sensitive calcium channels on [Ca2+]i in cultured frog adrenocortical cells. A–C, Typical profiles illustrating the effect of a single application of 10–6 M ET-1 in the absence (black traces) or presence (gray traces) of 10–6 M of the T-type calcium channel blocker mibefradil (A), 10–6 M of the N-type calcium channel blocker -conotoxin GVIA (B), or 10–6 M of the L-type calcium channel blocker nifedipine (C). Mibefradil, -conotoxin GVIA, and nifedipine were added to the incubation medium 30, 15, and 15 min before application of the pulse of ET-1, respectively. The arrows indicate the onset of ET-1 application. The histograms show that mibefradil and -conotoxin GVIA did not affect the [Ca2+]i response, whereas nifedipine reduced by 58.5% the AUC of the [Ca2+]i response induced by ET-1. NS, Not significantly different from the control; **, P < 0.01.
Involvement of Ca2+ in steroid secretion evoked by activation of ETA receptors
It has been previously shown that administration of repeated pulses of ET-1 causes a significant attenuation of the secretory response of perifused adrenocortical cells (22). To avoid this tachyphylaxis phenomenon, a single pulse of ET-1 (5 x 10–9 M) was administered to each perifusion experiment in the absence or presence of the Ca2+-ATPase inhibitor thapsigargin or the L-type Ca2+ channel blocker nifedipine. In control conditions, infusion of ET-1 (5 x 10–9 M; 20 min) provoked a marked increase in corticosterone and aldosterone secretion (Figs. 13, A and B, and 14, A and B). Prolonged infusion of thapsigargin (10–5 M) almost completely suppressed the stimulatory effect of ET-1 on corticosterone (Fig. 13C) and aldosterone production (Fig. 13D). In the presence of thapsigargin, the AUCs for corticosterone and aldosterone were reduced by 94.9 (P < 0.01; Fig. 13E) and 95.0% (P < 0.01; Fig. 13F), respectively, compared with the controls (Fig. 13, A and B). Infusion of nifedipine also provoked a significant inhibition of the effect of ET-1 on corticosterone (55.8%; P < 0.05; Fig. 14E) and aldosterone (61.6%; P < 0.05; Fig. 14F) secretion, respectively.
FIG. 13. Effect of ET-1 alone or during prolonged infusion of the calcium ATPase inhibitor thapsigargin on corticosteroid secretion by perifused frog adrenal slices. A and B, Control experiments showing the effect of ET-1 (5 x 10–9 M; 20 min) on corticosterone (A) and aldosterone (B) secretion. C and D, Effect of ET-1 during infusion of thapsigargin (10–5 M) on corticosterone (C) and aldosterone (D) secretion. E and F, The histograms show that thapsigargin reduced by 87.1 and 87.5% the AUCs of the corticosterone and aldosterone responses to ET-1, respectively. The arrows indicate the limits of the peaks that were used to calculate the AUCs. The profiles represent the mean (± SEM) secretion pattern of at least four independent experiments. Each point is the mean of the corticosteroid production (expressed as a percentage of spontaneous steroid output) in two consecutive fractions collected during 5 min. The mean basal levels of corticosterone and aldosterone secretion in these experiments were 66.7 ± 5.2 and 20.6 ± 1.8 pg/min per adrenal gland, respectively. **, P < 0.01.
FIG. 14. Effect of ET-1 alone or during prolonged infusion of the L-type calcium channel blocker nifedipine on corticosteroid secretion by perifused frog adrenal slices. A and B, Control experiments showing the effect of ET-1 (5 x 10–9 M; 20 min) on corticosterone (A) and aldosterone (B) secretion. C and D, Effect of ET-1 during infusion of nifedipine (10–5 M) on corticosterone (C) and aldosterone (D) secretion. E and F, The histograms show that nifedipine reduced by 48.9 and 58.9% the AUCs of the corticosterone and aldosterone responses to ET-1, respectively. The mean basal levels of corticosterone and aldosterone secretion in these experiments were 61.9 ± 7.1 and 13.7 ± 3.4 pg/min per adrenal gland, respectively. *, P < 0.05. See legend to Fig. 13 for other designations.
Discussion
We have previously reported that, in the frog adrenal gland, the stimulatory effect of ET-1 on corticosterone and aldosterone secretion is mediated by an ETA receptor (10, 22). We have also demonstrated the involvement of both the PLC and the AC signaling pathways in the response of frog adrenocortical cells to ET-1 (11). Here we show that ET-1 provokes Ca2+ mobilization from intracellular stores and Ca2+ influx through L-type Ca2+ channels. The data indicate that both sources of calcium are involved in ET-1-induced corticosteroid secretion.
Visualization of ET-1 receptors on adrenal cells
In contrast to mammals, the frog adrenal gland does not exhibit any zonation but is composed of intermingled steroidogenic and chromaffin tissue (23, 24). Therefore, cultures of frog adrenal cells are composed of a mixed population of adrenocortical and adrenochromaffin cells. However, the two types of cells can be easily distinguished on the basis of their typical morphological aspects: adrenocortical cells are spindle shaped with slender processes, whereas chromaffin cells are small and spherical (16). A cytoautoradiographic approach was applied to determine the cellular distribution of ET receptors in the frog adrenal gland. By using [125I]ET-1 as a radioligand, we found the presence of ET-1 binding sites on adrenocortical cells, whereas no labeling could be observed on chromaffin cells, suggesting a direct action of the peptide on corticosteroid secretion. Autoradiographic studies and binding experiments have revealed the same distribution of ET receptors in the human (25) and rat (26) adrenal gland: ETA receptors are present only in the zona glomerulosa, whereas ETB receptors occur throughout the entire gland.
Effect of ET-1 on [Ca2+]i
Consistent with the autoradiographic localization of ET-1 binding sites, an effect of ET-1 on Ca2+ transients was observed in 65% of adrenocortical cells but not in adrenochromaffin cells. Previous studies have shown that, in the frog adrenal gland, regulatory peptides such as ranakinin and pituitary AC-activating polypeptide exert an indirect corticotropic effect via activation of chromaffin cells (27, 28). The present study demonstrates that ET-1 induces a direct stimulatory effect on frog adrenocortical cells. Conversely, in mammals, it has been reported that ET-1 can exert both a direct effect on corticosteroid secretion (29) and an indirect effect through activation of chromaffin cell secretion (26). Application of ET-1 induced a dose-dependent increase of [Ca2+]i in frog adrenocortical cells with an EC50 value (1.7 x 10–9 M) that was in the same range as those previously described for ET-1-induced inositol phosphate production (1.3 x 10–9 M) (11) and ET-1-evoked corticosterone and aldosterone secretion (1.5 x 10–10 and 3 x 10–10 M, respectively) (3).
The ET-1-evoked [Ca2+]i transient was totally blocked by the ETA receptor antagonist BQ-485, indicating that the effects of ET-1 on both corticosteroid secretion (10) and [Ca2+]i increase are mediated through ETA receptors. In all responding cells, ET-1 provoked a biphasic increase in [Ca2+]i consisting of an immediate and transient peak followed by a plateau phase. A similar effect of ET-1 has previously been described in human (30), rat (31), and rabbit glomerulosa cells (29). The abrupt arrest of the plateau phase of the response usually observed 2–4 min after application of ET-1 can be ascribed either to ETA receptor internalization or to PKA inactivation. The fact that the PKA inhibitor H-89 suppressed the plateau phase of the Ca2+ wave would support the involvement of PKA inactivation in the arrest of the response. In two thirds of responding cells, ET-1 induced [Ca2+] oscillations, as already observed in bovine chondrocytes (32), rat gonadotrope cells (33), and rat pulmonary arterial (34) and preglomerular smooth muscle cells (35). The biphasic response and the oscillations induced by ET-1 suggested that both Ca2+ mobilization from intracellular stores and Ca2+ influx through membrane channels may contribute to the observed [Ca2+]i increase (36).
Involvement of intracellular calcium stores in the mechanism of action of ET-1
Exposure of adrenal cells to the PLC inhibitor U-73122 markedly reduced the amplitude of the early Ca2+ peak, suggesting that the transient response to ET-1 can be ascribed to mobilization of Ca2+ from inositol 1,4,5-triphosphate (IP3)-sensitive intracellular stores as previously described in other cell models (37, 38, 39). Consistent with this notion, depletion of the Ca2+ stores by thapsigargin, a Ca2+ ATPase inhibitor, reduced the amplitude of the transient phase of the ET-1-induced [Ca2+]i response. The contribution of intracellular Ca2+ stores to the [Ca2+]i response to ET-1 has recently been reported in rat glomerulosa cells (40). The observation that thapsigargin almost completely suppressed the stimulatory effect of ET-1 on corticosterone and aldosterone secretion from perifused frog adrenal slices suggests that intracellular Ca2+ from IP3-sensitive Ca2+ pools plays an important role in stimulus secretion coupling. Consistent with this notion, the involvement of IP3-induced Ca2+ mobilization from intracellular stores in the aldosterone response to ET-1 has recently been shown (40).
Involvement of calcium influx in the mechanism of action of ET-1
Suppression of Ca2+ in the incubation medium reduced the plateau phase of the [Ca2+]i response. Similarly, administration of the PKA inhibitor H-89 did not affect the early response but markedly attenuated the delayed phase of the [Ca2+]i wave, indicating the involvement of PKA-dependent calcium influx in the stimulatory effect of ET-1 on [Ca2+]i. The fact that both the transient peak and the plateau phase were almost suppressed by concomitant administration of U-73122 and H-89 is consistent with a dual effect of ET-1 on Ca2+ homeostasis in frog adrenocortical cells, i.e. an immediate increase in [Ca2+]i resulting from Ca2+ mobilization from intracellular stores and a delayed phase reflecting Ca2+ entry.
To determine which type of Ca2+ channel was responsible for the stimulatory effect of ET-1 on calcium influx and corticosteroid secretion, we used selective blockers of voltage-operated calcium channels. Preincubation of the cells with the L-type Ca2+ channel blocker nifedipine decreased the plateau phase of the [Ca2+]i response, whereas the T-type Ca2+ channel blocker mibefradil and the N-type Ca2+ channel blocker -conotoxin GVIA had no effect. In addition, exposure of frog adrenocortical cells to nifedipine reduced the stimulatory effect of ET-1 on corticosteroid secretion, indicating that the sustained phase of the [Ca2+]i increase is implicated in the secretory response. Interestingly, suppression of extracellular Ca2+ by EGTA and exposure of cells to nifedipine similarly attenuated the [Ca2+]i response and the corticosteroid burst induced by ET-1, confirming the contribution of Ca2+ influx in ET-1-evoked activation of adrenocortical cells. In agreement with these data, previous studies have shown the involvement of L-type Ca2+ channels in the stimulatory effect of ET-1 on rabbit (29), calf (41), and human adrenal cells (42). Concurrently, it should be noticed that, in frog, other corticotropic factors such as serotonin and triakontatetraneuropeptide provoke Ca2+ entry through activation of T-type Ca2+ channels (12, 13).
A proposed model illustrating the implication of calcium in the mechanism of action of ET-1 in frog adrenocortical cells is shown in Fig. 15. ET-1, acting via ETA receptors positively coupled to PLC, stimulates IP3 formation that provokes an immediate and transient release of Ca2+ from intracellular stores. Activation of ETA receptors also stimulates the AC-PKA pathway, which enhances the activity of L-type calcium channels. Calcium influx through these membrane channels is responsible for the plateau phase of the [Ca2+]i response. Both sources of calcium are involved in the stimulatory effect of ET-1 on corticosterone and aldosterone secretion.
FIG. 15. Proposed model depicting the mechanism of action of ET-1 in frog adrenocortical cells. Activation of ETA receptor causes stimulation of PLC and AC through heterotrimeric G proteins (G). The increase in IP3 formation triggers calcium mobilization from intracellular stores, whereas the stimulation of PKA activates L-type calcium channels, leading to an influx of calcium through the plasma membrane. Both sources of calcium are required to mediate the steroidogenic effect of ET-1 on corticosterone and aldosterone secretion.
Acknowledgments
The authors thank Huguette Lemonnier and Colette Piard for expert technical assistance.
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Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, European Institute for Peptide Research (Institut Fédratif de Recherches Multidisciplinaires sur les Peptides 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U 413, UA Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry@univ-rouen.fr.
Abstract
We have previously shown that endothelin (ET)-1 stimulates corticosterone and aldosterone secretion by the frog adrenal gland through activation of ETA receptors positively coupled to both the adenylyl cyclase and phospholipase C (PLC) pathways. The purpose of the present study was to investigate the involvement of calcium in ET-1-induced stimulation of corticosteroid secretion. Cytoautoradiographic labeling using [125I]ET-1 as a tracer revealed the presence of ET-1 binding sites on adrenocortical cells. Administration of graded concentrations of ET-1 in the vicinity of adrenocortical cells provoked a dose-dependent increase in cytosolic calcium concentrations ([Ca2+]i). ET-1 induced a biphasic response consisting of an immediate and transient peak of [Ca2+]i followed by a plateau phase. Preincubation of the cells with the calcium-ATPase inhibitor thapsigargin or the PLC inhibitor U-73122 reduced the amplitude of the transient phase. Administration of the calcium chelator EGTA or the protein kinase A inhibitor H-89 attenuated the plateau phase. The [Ca2+]i response to ET-1 was markedly reduced during concomitant administration of U-73122 and H-89. Preincubation of the cells with the L-type calcium channel blocker nifedipine attenuated the plateau phase. Corticosteroid secretion from perifused frog adrenal slices was almost completely suppressed by thapsigargin and reduced by nifedipine. Taken together, these data indicate that activation of ETA receptors in frog adrenocortical cells provokes immediate stimulation of PLC, which causes an early mobilization of calcium from intracellular stores, and activates adenylyl cyclase, which results in delayed calcium influx through L-type calcium channels. The resulting increase in [Ca2+]i plays a pivotal role in ET-1-induced corticosteroid secretion.
Introduction
THE ENDOTHELIN (ET) FAMILY comprises three isoforms of vasoactive peptides (ET-1, ET-2, and ET-3), which all possess 21 amino acids including four cysteine residues that form two intracellular disulfide bonds (1, 2). The primary structure of ETs has been strongly preserved during evolution. Notably, the sequence of ET-1 is identical in frog and human (3). The effects of ETs on smooth muscle cells are mediated through two types of G protein-coupled receptors that exhibit differential affinities for the isopeptides: the ETA receptor possesses higher affinity for ET-1 and ET-2 than for ET-3, whereas the ETB receptor has similar affinity for all three ET isoforms (4).
Besides the vascular bed, ET receptors are expressed in various endocrine and neuroendocrine tissues, and ETs modulate the secretion of a number of hormones (see Ref. 5 for review). In particular, ETs are potent activators of corticosteroid secretion in all vertebrate species investigated so far including human, bovine, rabbit, rat, and frog (6). However, the type of receptor mediating the effect of ETs on adrenocortical cells markedly differs from one species to the other. For instance, in the human adrenal gland, both ETA and ETB receptors are involved in the stimulatory effect of ET-1 on cortisol secretion (7). In rat, the stimulatory effect of ET-1 on fasciculata cells is mediated via ETB receptors (8), whereas the action of ET-1 on mineralocorticoid secretion has been ascribed to activation of either the ETB receptor only (8) or both the ETA and ETB receptors (9). In contrast, in frog, the effect of ET-1 on corticosteroid secretion is exclusively mediated through ETA receptors (10). The amphibian adrenal gland, which is composed of a single population of adrenocortical cells secreting both corticosterone and aldosterone, is a very suitable model in which to investigate the signaling mechanisms associated with activation of ETA receptors. A particular advantage of a pure population of endocrine cells, such as frog adrenocortical cells, in studying the transduction pathways downstream ETA receptors is that the effect of ET-1 on second messenger systems can be readily correlated with the final response of the cell by measuring corticosteroid secretion.
We previously demonstrated that, in frog adrenocortical cells, the stimulatory effect of ET-1 on corticosteroid secretion is mediated through activation of adenylyl cyclase (AC) and phospholipase C (PLC) (11). We have also shown that corticotropic agents stimulating AC can provoke calcium (Ca2+) influx through T-type Ca2+ channels (12, 13). Thus, in the present study, we investigated the effect of ETA receptor activation on calcium mobilization in frog adrenocortical cells in primary culture. The involvement of calcium in ET-1-induced corticosterone and aldosterone secretion has also been studied on perifused frog adrenal slices.
Materials and Methods
Reagents and test substances
Synthetic ET-1 (frog/human sequence) and ranakinin (frog substance P ortholog) were synthetized by the solid-phase methodology as previously described (14, 15). BQ-485 (perhydroazepin-L-yl-L-leucyl-D-tryptophanyl-D-tryptophan) was a generous gift from Banyu Pharmaceuticals (Tsukuba, Japan). Mibefradil (dihydrochloride) was kindly provided by Hoffmann-La Roche (Basel, Switzerland). The antiserum against 3?-hydroxysteroid dehydrogenase (3?-HSD) was generously provided by V. Luu-The (Laval University, Québec, Canada). Fluorescein isothiocyanate-conjugated goat antirabbit -globulins were purchased from Nordic Immunological Laboratories (Tilburg, The Netherlands). Leibovitz culture medium (L15), HEPES, thapsigargin, EGTA, collagenase (type IA), protease (from Bacillus polymyxa; type IX), nifedipine, -conotoxin GVIA, ionomycin, and BSA were obtained from Sigma (St. Louis, MO). Fetal bovine serum, the kanamycin solution (10,000 U/ml), and the antibiotic-antimycotic solution (penicillin G sodium, 10,000 U/ml; streptamycin sulfate, 10,000 U/ml) were from Life Technologies, Inc. (Grand Island, NY). 3-[125I]iodotyrosyl-ET-1 (2000 Ci/mmol), [1, 2, 6, 7-3H]corticosterone, and [1, 2, 6, 7-3H]aldosterone were from Amersham International (Les Ulis, France). Indo-1-pentacetoxymethylester was purchased from Molecular Probes (Leiden, The Netherlands). H-89, N-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinoline-sulfonamide, was from ICN Pharmaceuticals (Orsay, France). U-73122 [1-(6-[(17?-3-methoxyestra-1,3,5-(10)-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione] was from Biomol Research Laboratories (Plymouth Meeting, PA).
Animals
Adult male frogs (Rana esculenta; body weight 30–40 g) originating from Albania were obtained from a commercial supplier (Couétard, Saint-Hilaire de Riez, France). The animals were maintained in glass tanks supplied with a trickle of tap water, in a temperature-controlled room (8 ± 1 C) on a 12-h dark, 12-h light regimen (lights on from 0600 to 1800 h) for at least 1 wk before use. Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.
Cell culture
Frogs were killed by decapitation, and the adrenal glands were dissected free of renal parenchyma. The glands were washed three times with L15 medium adjusted to R. esculenta osmolality (L15/water = 1:0.4) supplemented with 0.4 mM CaCl2, 15 mM HEPES, 0.2 mM glucose, and 1% each of the kanamycin and antibiotic-antimycotic solutions (f-L15; pH 7.4). Adrenal cells were then enzymatically dispersed as previously described (16). Briefly, eight pairs of adrenal glands were subjected to enzymatic dissociation at 24 C for 45 min in an f-L15 solution 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. Dispersed cells were centrifuged (50 x g; 5 min) and rinsed three times with f-L15. The cells were suspended in 2 ml f-L15 medium supplemented with 10% fetal bovine serum and plated onto glass coverslips in petri dishes. The cells were kept at 22 C in an incubator with a humidified atmosphere. The culture medium was renewed 24 h after plating and subsequently every 48 h.
Immunofluorescence procedure
Immunohistochemical identification of adrenocortical cells (vs. chromaffin cells) was carried out on 3-d-old cells using the indirect immunofluorescence method, as previously described (16). The culture medium was aspirated and the cells were rinsed three times with 0.1 M phosphate buffer (PB; pH 7.4). The cells were fixed for 2 h at room temperature with a solution of 4% paraformaldehyde in PB and rinsed again three times with PB. The cells were then incubated 2 h at room temperature with the antiserum against 3?-HSD (1:100). The antiserum was diluted in PB with 1% BSA and 0.3% Triton X-100. The cells were rinsed with PB and incubated for 1 h at room temperature with fluorescein isothiocyanate-conjugated goat anti-rabbit -globulins (1:100). Finally, the cells were rinsed in PB, mounted with PB-glycerol (1:1), and coverslipped. The preparations were examined on a Orthoplan microscope (Leica, Heidelberg, Germany).
Cytoautoradiographic studies
Cells cultured for 3 d and plated at a low density (20,000 cells/cm2) were washed with 50 mM Tris buffer (pH 7.4) containing 0.2% BSA, 5 mM MgCl2, and 0.5 μg bacitracin per milliliter. The cells were incubated with 3-[125I]iodotyrosyl-ET-1 (100 pM) for 2 h at 24 C in Tris buffer supplemented with 0.2% BSA. Nonspecific binding was determined by adding 10–6 M ET-1. The coverslips were rinsed, glued to glass slides, and exposed to paraformaldehyde vapors (60 C for 24 h). The coverslips were then dipped into NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY) at 40 C. After 6 d of exposure, the cells were counterstained with Toluidine blue and examined under a Leitz Orthoplan microscope.
Calcium measurement
The effect of test substances on cytosolic calcium concentration ([Ca2+]i) was studied on single adrenocortical cells by a microfluorimetric technique, as previously described (16). Briefly, adrenal cells cultured for 3–5 d (70,000 cells/cm2) were incubated in darkness (24 C, 40 min) with 5 μM Indo-1-pentacetoxymethylester in f-L15 medium. The coverslips were washed with fresh medium and fitted to the stage of a Diaphot inverted microscope (Nikon, Melville, NY) equipped for epifluorescence with an oil immersion objective (x100 CF Fluor series; numerical operture 1.3). A pressure ejection system was used to deliver test substances in the vicinity of individual cells. 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 405:480 nm ratio R was determined using an analogic divider (constructed by Dr. B. Dufy, Bordeaux, France). All three signals (405 and 480 nm and R) were continuously recorded with a three-channel voltage recorder (BD 100/101, Kipp and Zonen, Delft, The Netherlands). [Ca2+]i was calculated from the formula established by Grynkiewicz et al. (17): [Ca2+]i = Kd x ? x [(R – Rmin)/(Rmax – R)], where Kd is the dissociation constant for indo-1 (250 nM), ? is the ratio of fluorescence yield from the Ca2+min/Ca2+max indicator at 480 nm, Rmin is the fluorescence ratio obtained after incubation of cells with f-L15 containing 10 mM EGTA and 10 μM ionomycin for 3 h, and Rmax is the fluorescence ratio obtained after incubation of cells with f-L15 containing 10 mM CaCl2 and 10 μM ionomycin for 3 h. The average values of ?, Rmin, and Rmax in frog adrenocortical cells were 1.77 (n = 20), 0.077 ± 0.008 (n = 15), and 1.033 ± 0.050 (n = 18), respectively.
Perifusion technique
The effect of test substances on corticosteroid secretion was studied by using a perifusion system technique, as previously described (10). For each perifusion experiment, six pairs of adrenal glands were sliced with scissors and preincubated in 5 ml Ringer’s solution (100 mM NaCl, 15 mM NaHCO3, 2 mM CaCl2, 2 mM KCl, 15 mM HEPES, 2 mg/ml glucose, and 0.3 mg/ml BSA). The Ringer’s solution was gassed with a 95% O2-5% CO2 mixture, and the pH was adjusted to 7.4. The adrenal slices were rinsed twice with Ringer’s medium and layered into a perifusion chamber between several beds of Bio-Gel P2 (Bio-Rad Laboratories, Ivry-sur-Seine, France). The adrenal slices were continuously perifused with gassed Ringer’s solution alone or with test substances, at constant flow rate (200 μl/min) and temperature (24 C). Fractions of effluent perfusate were collected every 5 min and frozen until assay. Nifedipine was dissolved in ethanol so that the final concentration of ethanol never exceeded 0.2%. Thapsigargin was dissolved in dimethylsulfoxide (DMSO) so that the final concentration of DMSO in the perifusion medium was 0.1%. Previous studies have shown that ethanol and DMSO, at concentrations of 0.2 and 0.1%, respectively, have no effect on corticosteroid secretion.
Corticosteroid RIA
Corticosterone and aldosterone concentrations were directly determined in 200- to 300-μl aliquots of each perifusion fraction without prior extraction, as previously described (18). Direct measurement of corticosterone and aldosterone has been validated by RIA quantification of corticosteroid after HPLC analysis of the effluent perfusate (19). The sensitivity thresholds of the assays were 20 pg for corticosterone and 5 pg for aldosterone. For both assays, the intra- and interassay coefficients of variation were 3 and 6% for corticosterone, and 5 and 8% for aldosterone, respectively. Each perifusion pattern was established as the mean profile of corticosteroid production (± SEM) calculated from at least three independent experiments. Corticosterone and aldosterone levels were expressed as percentages of the basal values, calculated as the mean of eight samples (40 min), taken just before the infusion of test substances.
Calculations
All data are expressed as mean ± SEM. The Mann-Whitney U test was used for comparison of the mean values between two groups. To compare the net increase in [Ca2+]i or steroid secretion induced by ET-1 in the absence or presence of various test substances, the areas under the curves (AUCs) were calculated using the trapezoidal rule (12).
Results
Identification of cultured adrenal cells
Under the light microscope, adrenocortical and chromaffin cells can be easily discriminated: the former are large, polygonal cells containing refractive lipid droplets, whereas the latter are spherical cells punctuated with secretory granules (Fig. 1A). Immunocytochemical labeling with antibodies against 3?-HSD confirmed the reliability of the morphological identification of adrenocortical cells (Fig. 1B).
FIG. 1. Micrographs of frog adrenal cells after 3 d of culture. A, Light photomicrograph showing that adrenocortical cells (arrows) are large cells containing refractive lipid droplets, whereas chromaffin cells (arrowhead) are spherical cells with punctuated granules. B, Immunocytochemical labeling of adrenocortical cells with antibodies against 3?-HSD. Bars, 10 μm.
Cellular localization of ET-1 binding sites
Cytoautoradiographic staining of cultured adrenal cells with [125I]ET-1 showed selective accumulation of silver grains on adrenocortical cells, whereas the density of grains on chromaffin cells was similar to that of the background (Fig. 2, A and B). In the presence of 10–6 M ET-1, autoradiographic labeling of adrenocortical cells was markedly reduced (Fig. 2, C and D).
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FIG. 2. Autoradiographic visualization of [125I]ET-1 binding sites on cultured frog adrenal cells. A, Photomicrograph of cultured cells stained with Toluidine blue, showing a dense accumulation of silver grains over adrenocortical cells (arrows); chromaffin cells were not labeled (arrowheads). B, Dark-field illumination micrograph of the field shown in A. C, Nonspecific binding in the presence of 10–6 M ET-1. D, Dark-field illumination micrograph of the field shown in C. Bars, 10 μm.
Effect of ET-1 on [Ca2+]i in cultured adrenocortical cells
Under resting conditions, the mean [Ca2+]i in cultured adrenocortical cells was 36.4 ± 2.1 nM (n = 328). Microejection of ET-1 (10–6 M; 5 sec) in the vicinity of adrenocortical cells provoked a marked increase in [Ca2+]i in 65% of the cells (n = 213), whereas application of vehicle had no effect (Fig. 3). In all responding cells, ET-1 evoked a biphasic response consisting of an immediate and transient peak of [Ca2+]i followed by a plateau phase (Fig. 3). Conversely, in chromaffin cells, ET-1 (10–6 M) has no effect on [Ca2+]i (n = 34), whereas the substance P analog ranakinin (10–6 M) elicited a robust increase in [Ca2+]i (Fig. 3, inset) as previously described (20). Application of graded concentrations of ET-1 (10–12 to 10–6 M) in the vicinity of adrenocortical cells produced a dose-dependent increase in [Ca2+]i with an EC50 of 1.7 x 10–9 M (Fig. 4). The minimum effective dose was 10–10 M (P < 0.05), and maximum stimulation was observed at a concentration of 10–7 M (P < 0.01). Recording of adrenocortical cells during 30 min (n = 46) revealed that ET-1 (10–6 M) induced Ca2+ oscillations in 65% of the responding cells (Fig. 5). The duration of each Ca2+ transient (81 ± 4 sec) and the mean frequency (8 x 10–3 Hz) were reproducible. In light of this oscillation phenomenon, each cell studied received a single dose of ET-1, and only one cell was recorded per petri dish. The selective ETA receptor antagonist BQ-485 (10–5 M) totally abolished the effect of ET-1 (10–6 M) on [Ca2+]i in adrenocortical cells (P < 0.001; Fig. 6).
FIG. 3. Typical profile illustrating the effect of a single application of 10–6 M ET-1 on [Ca2+]i in cultured frog adrenocortical cells. The profile is representative of 213 cells recorded, 65% of which responded to ET-1 by an increase in [Ca2+]i. Inset, ET-1 had no effect on [Ca2+]i in chromaffin cells, whereas 10–6 M ranakinin (RK), used as a control, induced a robust increase in [Ca2+]i. The arrows indicate the onset of application of culture medium alone (vehicle), ET-1, or RK.
FIG. 4. Dose-response curve showing the effect of graded concentrations of ET-1 on [Ca2+]i in cultured frog adrenocortical cells. The data have been calculated from a series of recordings similar to that presented in Fig. 3. The concentrations indicated on the x-axis are those contained in the ejection pipette. The number of cells studied at each point is indicated between parentheses. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG. 5. Effect of ET-1 (10–6 M) on [Ca2+]i oscillations in cultured frog adrenocortical cells. The profile is representative of 46 cells recorded, 65% of which responded to ET-1 by calcium transients. The arrows indicate the onset of vehicle or ET-1 application.
FIG. 6. Effect of ET-1 in the absence or presence of the selective ETA receptor antagonist BQ-485 (10–5 M) on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of 10–6 M ET-1. B, Incubation of cells with the ETA receptor antagonist BQ-485 totally suppressed the effect of ET-1 on [Ca2+]i. BQ-485 was added to the bath solution 60 min before application of the pulse of ET-1. The arrows indicate the onset of ET-1 application. The bar indicates the duration of BQ-485 administration. C, The histograms show that BQ-485 reduced by 99.3% the amplitude of the [Ca2+]i response induced by ET-1. ***, P < 0.001.
Sources of Ca2+ involved in ET-1-induced [Ca2+]i rise
Preincubation of the cells with the Ca2+ ATPase inhibitor thapsigargin (10–5 M; 5 min) induced a substantial elevation of [Ca2+]i from 28.9 ± 2.9 (n = 11) to 105.3 ± 9.5 nM (n = 13; Fig. 7, A and B). In the presence of thapsigargin, the amplitude of the early [Ca2+]i response to ET-1 was significantly reduced (P < 0.001; Fig. 7C). As a control, we have previously shown that thapsigargin does not impair serotonin-induced [Ca2+]i rise in frog adrenocortical cells (12). Concurrently, preincubation of the cells with the PLC inhibitor U-73122 (10–6 M; 60 min) did not affect basal [Ca2+]i but significantly attenuated (P < 0.01) the amplitude of the transient phase of ET-1-evoked Ca2+ increase (Fig. 8). As a control, it has been previously reported that U-73122 has no effect on serotonin-evoked [Ca2+]i increase in frog adrenocortical cells (12). Addition of 10 mM EGTA to f-L15, which reduced the concentration of free Ca2+ in the extracellular medium from 1.3 to 8 nM, provoked a significant reduction (P < 0.01) of the duration of the [Ca2+]i wave from 143 ± 17.9 to 80.1 ± 8.6 sec (Fig. 9, A and B). As a consequence, a 15-min incubation with EGTA significantly diminished (P < 0.01) the AUC of the [Ca2+]i response (Fig. 9C). Pilot experiments have shown that, in frog adrenocortical cells incubated with EGTA, thapsigargin could still provoke a robust increase in [Ca2+]i (Fig. 9B, inset), indicating that EGTA did not deplete the pool of intracellular Ca2+. In addition, we have previously shown that EGTA does not affect the response of frog adrenochromaffin cells to ranakinin (20).
FIG. 7. Effect of ET-1 in the absence or presence of the calcium ATPase inhibitor thapsigargin on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of 10–6 M ET-1. B, Effect of ET-1 in the presence of 10–5 M thapsigargin. Thapsigargin was added to the bath solution 5 min before application of the pulse of ET-1. The arrows indicate the onset of vehicle or ET-1 application. The bar indicates the duration of thapsigargin administration. C, The histograms show that thapsigargin reduced by 60.9% the amplitude of the [Ca2+]i response induced by ET-1. ***, P < 0.001.
FIG. 8. Effect of ET-1 in the absence or presence of the PLC inhibitor U-73122 on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of 10–6 M ET-1. B, Effect of ET-1 in the presence of 10–6 M U-73122. U-73122 was added to the bath solution 60 min before application of the pulse of ET-1. The arrows indicate the onset of ET-1 application. The bar indicates the duration of U-73122 administration. C, The histograms show that U-73122 reduced by 73.1% the amplitude of the [Ca2+]i response induced by ET-1. **, P < 0.01.
FIG. 9. Effect of ET-1 in the absence or presence of the Ca2+ chelator EGTA on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of 10–6 M ET-1. B, Effect of ET-1 in the presence of 10–2 M EGTA. EGTA was added to the bath solution 5 min before application of the pulse of ET-1. Inset, EGTA did not deplete the pool of intracellular Ca2+ because thapsigargin (Tg) could still provoke a massive increase in [Ca2+]i. The arrows indicate the onset of ET-1 application. The bar indicates the duration of EGTA administration. C, The histograms show that EGTA reduced by 55.1% the AUC of the [Ca2+]i response induced by ET-1. **, P < 0.01.
Administration of the protein kinase A (PKA) inhibitor H-89 (10–5 M) did not affect the amplitude of the early [Ca2+]i peak but totally suppressed the delayed phase (Fig. 10, A and B). A brief ejection of H-89 during the plateau phase provoked a rapid decrease of [Ca2+]i to basal level (Fig. 10C). During prolonged incubation with H-89, the AUC of the [Ca2+]i response was significantly reduced (P < 0.001; Fig. 10D). Concomitant incubation of adrenocortical cells with U-73122 (10–6 M) and H-89 (10–5 M) almost completely suppressed the [Ca2+]i response induced by ET-1 (P < 0.001; Fig. 11). Preincubation of adrenocortical cells with the T-type Ca2+ channel blocker mibefradil (10–6 M; 15 min; Fig. 12A) or the N-type Ca2+ channel blocker -conotoxin GVIA (10–6 M; 30 min; Fig. 12B) did not modify the [Ca2+]i response evoked by ET-1. As controls, we have previously shown that mibefradil markedly attenuates triakontatetraneuropeptide-induced [Ca2+]i rise in frog adrenocortical cells (13) and that -conotoxin GVIA reduces neurotensin-evoked [Ca2+]i rise in frog melanotrope cells (21). Conversely, the L-type channel blocker nifedipine (10–6 M; 15 min) diminished the amplitude and duration of the plateau phase without affecting the amplitude of the transient phase (Fig. 12C). Thus, nifedipine significantly reduced the AUC of the [Ca2+]i response (P < 0.01; Fig. 12C).
FIG. 10. Effect of ET-1 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 10–6 M ET-1. B and C, Effect of ET-1 in the presence of 10–5 M H-89. B, H-89 was added to the bath solution 60 min before application of the pulse of ET-1. C, H-89 was ejected during the plateau phase of the response. The arrows indicate the onset of ET-1 or H-89 application. The bar indicates the duration of H-89 administration. D, The histograms show that H-89 reduced by 75.7% the AUC of the [Ca2+]i response induced by ET-1. ***, P < 0.001.
FIG. 11. Effect of ET-1 in the absence or presence of the PLC inhibitor U-73122 and the PKA inhibitor H-89 on [Ca2+]i in cultured frog adrenocortical cells. A, Typical profile illustrating the effect of a single application of 10–6 M ET-1. B, Effect of ET-1 in the presence of 10–6 M U-73122 plus 10–5 M H-89. U-73122 and H-89 were added to the bath solution 60 min before application of the pulse of ET-1. The arrows indicate the onset of ET-1 application. The bar indicates the duration of U-73122 and H-89 administration. C, The histograms show that concomitant administration of U-73122 and H-89 reduced by 89.9% the amplitude of the [Ca2+]i response induced by ET-1. ***, P < 0.001.
FIG. 12. Effect of ET-1 in the absence or presence of selective blockers of voltage-sensitive calcium channels on [Ca2+]i in cultured frog adrenocortical cells. A–C, Typical profiles illustrating the effect of a single application of 10–6 M ET-1 in the absence (black traces) or presence (gray traces) of 10–6 M of the T-type calcium channel blocker mibefradil (A), 10–6 M of the N-type calcium channel blocker -conotoxin GVIA (B), or 10–6 M of the L-type calcium channel blocker nifedipine (C). Mibefradil, -conotoxin GVIA, and nifedipine were added to the incubation medium 30, 15, and 15 min before application of the pulse of ET-1, respectively. The arrows indicate the onset of ET-1 application. The histograms show that mibefradil and -conotoxin GVIA did not affect the [Ca2+]i response, whereas nifedipine reduced by 58.5% the AUC of the [Ca2+]i response induced by ET-1. NS, Not significantly different from the control; **, P < 0.01.
Involvement of Ca2+ in steroid secretion evoked by activation of ETA receptors
It has been previously shown that administration of repeated pulses of ET-1 causes a significant attenuation of the secretory response of perifused adrenocortical cells (22). To avoid this tachyphylaxis phenomenon, a single pulse of ET-1 (5 x 10–9 M) was administered to each perifusion experiment in the absence or presence of the Ca2+-ATPase inhibitor thapsigargin or the L-type Ca2+ channel blocker nifedipine. In control conditions, infusion of ET-1 (5 x 10–9 M; 20 min) provoked a marked increase in corticosterone and aldosterone secretion (Figs. 13, A and B, and 14, A and B). Prolonged infusion of thapsigargin (10–5 M) almost completely suppressed the stimulatory effect of ET-1 on corticosterone (Fig. 13C) and aldosterone production (Fig. 13D). In the presence of thapsigargin, the AUCs for corticosterone and aldosterone were reduced by 94.9 (P < 0.01; Fig. 13E) and 95.0% (P < 0.01; Fig. 13F), respectively, compared with the controls (Fig. 13, A and B). Infusion of nifedipine also provoked a significant inhibition of the effect of ET-1 on corticosterone (55.8%; P < 0.05; Fig. 14E) and aldosterone (61.6%; P < 0.05; Fig. 14F) secretion, respectively.
FIG. 13. Effect of ET-1 alone or during prolonged infusion of the calcium ATPase inhibitor thapsigargin on corticosteroid secretion by perifused frog adrenal slices. A and B, Control experiments showing the effect of ET-1 (5 x 10–9 M; 20 min) on corticosterone (A) and aldosterone (B) secretion. C and D, Effect of ET-1 during infusion of thapsigargin (10–5 M) on corticosterone (C) and aldosterone (D) secretion. E and F, The histograms show that thapsigargin reduced by 87.1 and 87.5% the AUCs of the corticosterone and aldosterone responses to ET-1, respectively. The arrows indicate the limits of the peaks that were used to calculate the AUCs. The profiles represent the mean (± SEM) secretion pattern of at least four independent experiments. Each point is the mean of the corticosteroid production (expressed as a percentage of spontaneous steroid output) in two consecutive fractions collected during 5 min. The mean basal levels of corticosterone and aldosterone secretion in these experiments were 66.7 ± 5.2 and 20.6 ± 1.8 pg/min per adrenal gland, respectively. **, P < 0.01.
FIG. 14. Effect of ET-1 alone or during prolonged infusion of the L-type calcium channel blocker nifedipine on corticosteroid secretion by perifused frog adrenal slices. A and B, Control experiments showing the effect of ET-1 (5 x 10–9 M; 20 min) on corticosterone (A) and aldosterone (B) secretion. C and D, Effect of ET-1 during infusion of nifedipine (10–5 M) on corticosterone (C) and aldosterone (D) secretion. E and F, The histograms show that nifedipine reduced by 48.9 and 58.9% the AUCs of the corticosterone and aldosterone responses to ET-1, respectively. The mean basal levels of corticosterone and aldosterone secretion in these experiments were 61.9 ± 7.1 and 13.7 ± 3.4 pg/min per adrenal gland, respectively. *, P < 0.05. See legend to Fig. 13 for other designations.
Discussion
We have previously reported that, in the frog adrenal gland, the stimulatory effect of ET-1 on corticosterone and aldosterone secretion is mediated by an ETA receptor (10, 22). We have also demonstrated the involvement of both the PLC and the AC signaling pathways in the response of frog adrenocortical cells to ET-1 (11). Here we show that ET-1 provokes Ca2+ mobilization from intracellular stores and Ca2+ influx through L-type Ca2+ channels. The data indicate that both sources of calcium are involved in ET-1-induced corticosteroid secretion.
Visualization of ET-1 receptors on adrenal cells
In contrast to mammals, the frog adrenal gland does not exhibit any zonation but is composed of intermingled steroidogenic and chromaffin tissue (23, 24). Therefore, cultures of frog adrenal cells are composed of a mixed population of adrenocortical and adrenochromaffin cells. However, the two types of cells can be easily distinguished on the basis of their typical morphological aspects: adrenocortical cells are spindle shaped with slender processes, whereas chromaffin cells are small and spherical (16). A cytoautoradiographic approach was applied to determine the cellular distribution of ET receptors in the frog adrenal gland. By using [125I]ET-1 as a radioligand, we found the presence of ET-1 binding sites on adrenocortical cells, whereas no labeling could be observed on chromaffin cells, suggesting a direct action of the peptide on corticosteroid secretion. Autoradiographic studies and binding experiments have revealed the same distribution of ET receptors in the human (25) and rat (26) adrenal gland: ETA receptors are present only in the zona glomerulosa, whereas ETB receptors occur throughout the entire gland.
Effect of ET-1 on [Ca2+]i
Consistent with the autoradiographic localization of ET-1 binding sites, an effect of ET-1 on Ca2+ transients was observed in 65% of adrenocortical cells but not in adrenochromaffin cells. Previous studies have shown that, in the frog adrenal gland, regulatory peptides such as ranakinin and pituitary AC-activating polypeptide exert an indirect corticotropic effect via activation of chromaffin cells (27, 28). The present study demonstrates that ET-1 induces a direct stimulatory effect on frog adrenocortical cells. Conversely, in mammals, it has been reported that ET-1 can exert both a direct effect on corticosteroid secretion (29) and an indirect effect through activation of chromaffin cell secretion (26). Application of ET-1 induced a dose-dependent increase of [Ca2+]i in frog adrenocortical cells with an EC50 value (1.7 x 10–9 M) that was in the same range as those previously described for ET-1-induced inositol phosphate production (1.3 x 10–9 M) (11) and ET-1-evoked corticosterone and aldosterone secretion (1.5 x 10–10 and 3 x 10–10 M, respectively) (3).
The ET-1-evoked [Ca2+]i transient was totally blocked by the ETA receptor antagonist BQ-485, indicating that the effects of ET-1 on both corticosteroid secretion (10) and [Ca2+]i increase are mediated through ETA receptors. In all responding cells, ET-1 provoked a biphasic increase in [Ca2+]i consisting of an immediate and transient peak followed by a plateau phase. A similar effect of ET-1 has previously been described in human (30), rat (31), and rabbit glomerulosa cells (29). The abrupt arrest of the plateau phase of the response usually observed 2–4 min after application of ET-1 can be ascribed either to ETA receptor internalization or to PKA inactivation. The fact that the PKA inhibitor H-89 suppressed the plateau phase of the Ca2+ wave would support the involvement of PKA inactivation in the arrest of the response. In two thirds of responding cells, ET-1 induced [Ca2+] oscillations, as already observed in bovine chondrocytes (32), rat gonadotrope cells (33), and rat pulmonary arterial (34) and preglomerular smooth muscle cells (35). The biphasic response and the oscillations induced by ET-1 suggested that both Ca2+ mobilization from intracellular stores and Ca2+ influx through membrane channels may contribute to the observed [Ca2+]i increase (36).
Involvement of intracellular calcium stores in the mechanism of action of ET-1
Exposure of adrenal cells to the PLC inhibitor U-73122 markedly reduced the amplitude of the early Ca2+ peak, suggesting that the transient response to ET-1 can be ascribed to mobilization of Ca2+ from inositol 1,4,5-triphosphate (IP3)-sensitive intracellular stores as previously described in other cell models (37, 38, 39). Consistent with this notion, depletion of the Ca2+ stores by thapsigargin, a Ca2+ ATPase inhibitor, reduced the amplitude of the transient phase of the ET-1-induced [Ca2+]i response. The contribution of intracellular Ca2+ stores to the [Ca2+]i response to ET-1 has recently been reported in rat glomerulosa cells (40). The observation that thapsigargin almost completely suppressed the stimulatory effect of ET-1 on corticosterone and aldosterone secretion from perifused frog adrenal slices suggests that intracellular Ca2+ from IP3-sensitive Ca2+ pools plays an important role in stimulus secretion coupling. Consistent with this notion, the involvement of IP3-induced Ca2+ mobilization from intracellular stores in the aldosterone response to ET-1 has recently been shown (40).
Involvement of calcium influx in the mechanism of action of ET-1
Suppression of Ca2+ in the incubation medium reduced the plateau phase of the [Ca2+]i response. Similarly, administration of the PKA inhibitor H-89 did not affect the early response but markedly attenuated the delayed phase of the [Ca2+]i wave, indicating the involvement of PKA-dependent calcium influx in the stimulatory effect of ET-1 on [Ca2+]i. The fact that both the transient peak and the plateau phase were almost suppressed by concomitant administration of U-73122 and H-89 is consistent with a dual effect of ET-1 on Ca2+ homeostasis in frog adrenocortical cells, i.e. an immediate increase in [Ca2+]i resulting from Ca2+ mobilization from intracellular stores and a delayed phase reflecting Ca2+ entry.
To determine which type of Ca2+ channel was responsible for the stimulatory effect of ET-1 on calcium influx and corticosteroid secretion, we used selective blockers of voltage-operated calcium channels. Preincubation of the cells with the L-type Ca2+ channel blocker nifedipine decreased the plateau phase of the [Ca2+]i response, whereas the T-type Ca2+ channel blocker mibefradil and the N-type Ca2+ channel blocker -conotoxin GVIA had no effect. In addition, exposure of frog adrenocortical cells to nifedipine reduced the stimulatory effect of ET-1 on corticosteroid secretion, indicating that the sustained phase of the [Ca2+]i increase is implicated in the secretory response. Interestingly, suppression of extracellular Ca2+ by EGTA and exposure of cells to nifedipine similarly attenuated the [Ca2+]i response and the corticosteroid burst induced by ET-1, confirming the contribution of Ca2+ influx in ET-1-evoked activation of adrenocortical cells. In agreement with these data, previous studies have shown the involvement of L-type Ca2+ channels in the stimulatory effect of ET-1 on rabbit (29), calf (41), and human adrenal cells (42). Concurrently, it should be noticed that, in frog, other corticotropic factors such as serotonin and triakontatetraneuropeptide provoke Ca2+ entry through activation of T-type Ca2+ channels (12, 13).
A proposed model illustrating the implication of calcium in the mechanism of action of ET-1 in frog adrenocortical cells is shown in Fig. 15. ET-1, acting via ETA receptors positively coupled to PLC, stimulates IP3 formation that provokes an immediate and transient release of Ca2+ from intracellular stores. Activation of ETA receptors also stimulates the AC-PKA pathway, which enhances the activity of L-type calcium channels. Calcium influx through these membrane channels is responsible for the plateau phase of the [Ca2+]i response. Both sources of calcium are involved in the stimulatory effect of ET-1 on corticosterone and aldosterone secretion.
FIG. 15. Proposed model depicting the mechanism of action of ET-1 in frog adrenocortical cells. Activation of ETA receptor causes stimulation of PLC and AC through heterotrimeric G proteins (G). The increase in IP3 formation triggers calcium mobilization from intracellular stores, whereas the stimulation of PKA activates L-type calcium channels, leading to an influx of calcium through the plasma membrane. Both sources of calcium are required to mediate the steroidogenic effect of ET-1 on corticosterone and aldosterone secretion.
Acknowledgments
The authors thank Huguette Lemonnier and Colette Piard for expert technical assistance.
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