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Expression and Regulation of Adenylyl Cyclase Isoforms in the Human Adrenal Gland

Service of Endocrinology, Department of Medicine (M.C., N.G.-P.), and Department of Physiology and Biophysics (M.C., M.D.P., N.G.-P.), Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4; and INSERM U469 (G.G.), Rue de la Cardonille, 34094 Montpellier Cedex, France

Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Department of Medicine, Faculty of Medicine, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4. E-mail: ngallo01{at}courrier.usherb.ca

Abstract

The aim of the present study was to identify which adenylyl cyclase isoforms were expressed in the human adrenal gland and to determine which isoform(s) may be coupled to ACTH action. Our results indicate that, in both glomerulosa and fasciculata zones, adenylyl cyclase 1 was detected in cells at the membrane level, adenylyl cyclases 3 and 2 in both the cytoplasm and the plasma membrane, whereas adenylyl cyclase 5/6 and adenylyl cyclase 4 were found mainly in cytoplasm. The levels of expression of each isoform were similar between the two adrenocortical zones, except for adenylyl cyclase 5/6, which had a lower level of expression in the zona fasciculata. We next evaluated the role of the various adenylyl cyclase isoforms during ACTH-stimulated cAMP production in both glomerulosa and fasciculata cell preparations. Corroborating with previous observations, we found that calcium had a biphasic effect on cAMP production. Interestingly, pertussis toxin treatment increased cAMP production, indicating that, in addition to Gs, ACTH is coupled to a Gi protein. Incubation with the ß-subunit sequestrant peptide QEHA decreased cAMP production, as did incubation with inhibitory antibodies against either adenylyl cyclase 2 or adenylyl cyclase 5/6. Inhibitory adenylyl cyclase 3 antibodies interfered with ACTH action only in the zona fasciculata. Altogether these data indicate that adrenocortical cells express one or two isoforms of each class of adenylyl cyclases and, thus, have the ability to produce cAMP in response to various regulatory, intracellular mediators. Importantly, our results indicate that in the human adrenal gland, ACTH acts mainly through adenylyl cyclase 5/6 and adenylyl cyclase 2/4, whereas the effect of ACTH on adenylyl cyclase 3 activity may be a consequence of calcium influx.

SEVERAL PIECES OF evidence indicate that adrenocortical steroidogenesis is modulated not only by ACTH, but also by a variety of neuropeptides and neurotransmitters (1). Among the important list of stimuli that regulate aldosterone and cortisol secretion via the cAMP-PKA pathway, adrenocorticotropic hormone (ACTH) remains the most potent stimulus of cAMP production causing increases ranging from 20- to 40-fold, depending on the model or conditions studied. ACTH first activates the conversion of cholesterol to pregnenolone and, subsequently, to cortisol in fasciculata cells and to 18-hydroxy-corticosterone and aldosterone in glomerulosa cells. As a more long-term effect, ACTH stimulates biosynthesis of the enzymes involved in steroidogenesis by increasing their respective mRNA levels (2). ACTH also has an important trophic action on adrenal gland architecture (3). It is well known that hypophysectomy decreases, whereas ACTH treatment or Cushing’s disease increases, adrenal gland volume. The action of ACTH occurs through the binding its specific receptor, causing an increase in intracellular cAMP levels, followed by the activation of PKA. Despite several studies using both animal and human glomerulosa or fasciculata cells, the precise molecular mechanisms by which ACTH stimulates steroid synthesis and secretion is still poorly understood. Since the pioneering work of Lefkowitz et al. (4) in 1970, several studies have shown that cAMP and Ca²⁺ closely interact through positive feedback loops to enhance steroid secretion (5, 6, 7). We have recently shown that ACTH, in human adrenocortical cells, not only controls cAMP production, but also cAMP degradation, acting directly on phosphodiesterase activity (8).

In addition to these intracellular interactions, one of the primary effectors identified able to explain the sensitivity of adrenocortical cells to ACTH stimulation may well be adenylyl cyclase (AC) . Nine isoforms of ACs have been cloned and grouped into four classes (9, 10, 11, 12). The regulation of AC by G protein-coupled receptors is a classically described mechanism in which a stimulatory G protein (G_s) is coupled to activation of AC, whereas an inhibitory G protein (G_i) is coupled to inhibition of AC. ACs demonstrate diverse regulatory properties in the control of cAMP production. AC1, AC3, and AC8 are sensitive to Ca²⁺ and calmodulin and are inhibited by _i-subunits; AC2, AC4, and AC7 are stimulated by ß-subunits of the heterotrimeric G proteins and PKC; AC5 and AC6 are inhibited by Ca²⁺ and PKA; AC9 is inhibited by Ca²⁺, but its regulation is still under debate (9, 10, 11, 12). Regulation of ACs by Ca²⁺, calmodulin, G protein ß-subunits, PKC, or PKA is a proposed mechanism for positive or negative feedback loops, which could ultimately affect specific cortisol or aldosterone secretion under ACTH stimulation. The observation that Ca²⁺ greatly influences the potency of ACTH in stimulating cAMP production (7, 13, 14) and our recent findings that, in rat glomerulosa cells, ACTH acts not only through s, but also through G protein ß-subunits (15) suggest that various AC isoforms could be expressed in the adrenal cortex. Presently, there is no information regarding the expression of AC isoforms in the human adrenal gland or their modulation following ACTH stimulation. Therefore, the aim of the present study was to identify the nature of ACs present in human glomerulosa vs. fasciculata cells and to study their functional regulation.

Materials and Methods

Chemicals

The chemicals used in the present study were obtained from the following sources: [³²P]-ATP (3000 Ci/mmol), [2,8-³H]-cAMP (24 Ci/mmol), and horseradish peroxidase-conjugated antirabbit antibody from Amersham Pharmacia Biotech (Oakville, Canada); ATP, cAMP, EDTA, dithiothreitol, pertussis toxin (PTX), and DNase from Sigma (St. Louis, MO); ACTH 1–24 peptide (Cortrosyn) from Organon (Toronto, Canada); creatine kinase, creatine phosphate disodium, EGTA, enhanced chemiluminescence detection system, and FITC from Roche Molecular Biochemicals (Montreal, Quebec, Canada); AC1 to AC8 antibodies from Santa Cruz Biotechnology, Inc. (La Jolla, CA); H-89 and GF109203x from Calbiochem-Novabiochem (La Jolla, CA); collagenase, MEM Eagle medium, and OPTI-MEM medium from Life Technologies, Inc. (Burlington, Canada); Vectastain Elite ABC Reagent and VectaMount from Vector Laboratories, Inc. (Burlingame, CA). The QEHA and SKEE peptides, which have 23 and 32 amino acids, respectively (16), were synthesized, purified, and identified by mass spectrometry by the Service Protéomique de l’Est du Québec (Le Center de Recherche du Center Hospitalier de l’Université Laval, Quebec, Canada). All other chemicals were of grade-A purity.

Immunohistochemical studies

Human adrenal glands were obtained from renal transplant donors, 16–22 yr old, through collaboration with the Quebec-Transplant Association. The Human Subject Review Committee of our institution approved this project. After removal, glands were immersed immediately in 4% paraformaldehyde, embedded in paraffin, cut into 3-µm sections, and processed by indirect immunoperoxidase immunohistochemistry. The sections were incubated in 0.3% H₂O₂ to quench endogenous peroxidase activity, then incubated with 250 ng/ml rabbit polyclonal antibodies against AC1 to AC8, for 18 h at 4 C. Where indicated, peptide antigens were added in 20-fold molar excess to the antibody immediately before incubation. Sections were incubated with rabbit-biotinylated secondary antibody, then washed and incubated with Vectastain Elite ABC Reagent, followed by detection using the 3,3'-diaminobenzidine reaction. Counterstaining was performed using hematoxylin, and the slides were mounted in nonaqueous mounting medium, VectaMount. Immunolabeling was observed using a Nikon Eclipse 300 microscope (Nikon Canada Inc., Mississauga, Ontario, Canada) equipped with a CoolSnap color digital camera. Acquired images were processed by Adobe Photoshop 4.0 (Microsoft).

Preparation of adrenocortical cells

After removal, glands were kept on ice in McCoy’s medium, transported within 4 h to the laboratory, and processed as described previously (7, 17). Capsule and zona glomerulosa (first slice) were used to prepare glomerulosa cells, whereas the remaining cortex was used to prepare the so-called fasciculata cells. The cells were cultured in Eagle’s MEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified atmosphere of 95% air/5% CO₂. The culture medium was changed daily, and the cells were used after 3 d of culture where cell density was approximately 3.0 x 10⁵ cells/dish.

Triton-insoluble fraction preparation

Culture medium was aspirated and changed for HBSS buffer. Cells were collected and centrifuged at 100 x g. One hundred microliters of Triton solution [1% Triton X-100, 10 mM EGTA, 0.1 M Tris-HCl (pH 7.4)] were added and the solution was transferred to 1.5-ml microcentrifuge tubes and centrifuged at 8000 x g. The pellet, corresponding to the Triton-insoluble fraction, was solubilized in 2% SDS + 2% 2ß-mercaptoethanol (vol/vol), was aliquoted and frozen for subsequent Western blot analysis (18).

Membrane preparation

Membranes were prepared as described previously (8). Zona glomerulosa or fasciculata was homogenized with a Polytron homogenizer in cold buffer containing 50 mM Tris-HCl (pH 7.6), 0.1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonylfluoride and centrifuged at 150 x g for 10 min, and the supernatant was recentrifuged at 15,000 x g for 30 min. The crude membrane fraction was washed twice in the same buffer and frozen at -80 C for subsequent analysis.

AC activity

Membranes were incubated for 10 min at 37 C in a 60-µl reaction volume containing 50 mM Tris-HCl (pH 7.6), 1 mM MgCl₂, 0.04 mM GTP, 1 nM free-Ca²⁺, 0.1 mM cAMP, 0.1 mM ATP (containing 1 µCi [³²P]ATP), 0.25 mg/ml creatine kinase, and 1.3 mg/ml creatine phosphate, with or without either 10 pM and 10 nM ACTH and the compounds to be tested (100 ng/ml cyclase antibody and 100 µM QEHA or 100 µM SKEE peptide preincubated 10 min before addition of ACTH) (8, 19). [³H]-cAMP (10,000 cpm) was used as an internal standard to measure overall recovery. All experiments were conducted in the presence of 1 mM Mg²⁺, a concentration sufficient for catalysis of ATP, but which does not interfere with the Ca²⁺-dependent sensitivity of AC (20). For all experiments, the Chelate computer program was used to calculate the concentration of free Ca²⁺ in EGTA-buffered incubation medium. Protein concentration was determined according to the Bradford method.

cAMP accumulation

Intracellular cAMP production was determined by measuring the conversion of [³H]-ATP to [³H]- cAMP, as described previously (13). In short, cultured cells were incubated at 37 C in OPTI-MEM culture medium containing 2 µCi/ml [³H]-adenine. After 1 h the cultures were washed and incubated with hormones or drugs in a HBSS buffer containing 1 mM isobutylmethylxanthine. After 15 min at 37 C, cells were collected, solubilized, and chromatographed on Dowex and alumina columns according to the method of Salomon et al. (21).

Western blotting

Samples from equivalent amounts of proteins were compared in each experiment and separated on 12% SDS-polyacrylamide gels. Proteins were transferred electrophoretically to nitrocellulose. Membranes were blocked with 5% fat-free milk and 0.05% Tween 20 in Tris-buffered saline (TBS) (pH 7.4). After three washes with TBS-Tween 20 (0.05%), membranes were incubated with one of the anti-AC isoforms to be tested (AC1 to AC5/6, 1:200) overnight at 4 C, followed by four washes with TBS-Tween 20. For stripping, membranes were incubated in 50 mM Tris, 2% SDS, and 0.7% ß-mercaptoethanol, followed by five washes in TBS-Tween 20 before reincubation with another antibody. Detection was accomplished with horseradish peroxidase-conjugated antimouse antibody and an enhanced chemiluminescence detection system.

Data analysis

The data are presented as means ± SEM. Statistical analyses of the data were performed using the one-way ANOVA test. Homogeneity of variance was assessed by Bartlett’s test and P values were obtained from Dunnett’s tables. n indicates the number of experiments, each performed in duplicate or triplicate.

Results

Localization and expression of AC proteins in the human adrenal gland

The distribution and the cellular localization of the Ca²⁺-sensitive AC isoforms were detected by immunoperoxidase immunohistochemistry, using specific antibodies against each of the different AC isoforms. The AC1 antibody produced an intense labeling at the membrane level in both the zona glomerulosa (Fig. 1A) and the zona fasciculata (Fig. 1B). The AC3 isoform was also detected in the two adrenocortical cell types. In contrast to AC1, AC3 was detected not only at the membrane but also in the cytoplasm, surrounding lipid droplets (Fig. 1, D and E). As shown in Fig. 1, C and F, labeling was completely abrogated after AC1 and AC3 peptide adsorption. The Ca²⁺-inhibitable AC5/AC6 isoforms, detectable with the same antibody, were concentrated in the cytoplasm rather than in the membrane (Fig. 2, A and B) and was more abundantly found in the zona glomerulosa (Fig. 2A) than in the zona fasciculata (Fig. 2B). Figure 2C shows that the labeling was completely abrogated after AC5/AC6 peptide adsorption. Localization of the ß- and PKC-sensitive isoform, AC2 was observed both at the membrane level and in the cytoplasm and was expressed with the same intensity in both the zona glomerulosa and the zona fasciculata (Fig. 3, A and B). In contrast, AC4 labeling was concentrated in the cytoplasm of both zones (Fig. 3, D and E). Again, labeling was completely abrogated after AC2 and AC4 peptide adsorption (Fig. 3, C and F). Two isoforms, AC7 and AC8, were not detected (data not shown).

View larger version (170K): [in this window] [in a new window]   Figure 1. Immunohistochemical localization of the Ca2+-sensitive AC isoforms AC1 and AC3 in the human adrenal gland. Whole gland sections were processed for immunohistochemical localization as described in Materials and Methods. A, AC1, zona glomerulosa (objective, x100). B, AC1, zona fasciculata (objective, x100). C, peptide adsorption of AC1 antibody by AC1 peptide (objective, x100). D, AC3, zona glomerulosa (objective, x100). E, AC3, zona fasciculata (objective, x100). F, Peptide adsorption of AC3 antibody by AC3 peptide (objective, x100). The images are representative illustrations of three different human adrenal glands. Scale bars, 20 µm.

View larger version (87K): [in this window] [in a new window]   Figure 2. Immunohistochemical localization of the Ca2+-inhibitable AC AC5/AC6 isoform in the human adrenal gland. Whole gland sections processed for immunohistochemical localization as described in Materials and Methods. A, zona glomerulosa (objective, x100). B, zona fasciculata (objective, x100). C, peptide adsorption of AC5/AC6 antibody by AC5/AC6 peptide (objective, x100). The images are representative illustrations of three different adrenal glands. Scale bars, 20 µm.

View larger version (172K): [in this window] [in a new window]   Figure 3. Immunohistochemical localization of the ß- and PKC-sensitive AC isoforms AC2 and AC4 in the human adrenal gland. Whole gland sections were processed for immunohistochemical localization as described in Materials and Methods. Zona glomerulosa for AC2 (A) and AC4 (D) (objective, x100); zona fasciculata for AC2 (B) and AC4 (E) (objective, x100); peptide adsorption of AC2 antibody by AC2 peptide (C) and AC4 antibody by AC4 peptide (F) (objective, x100). The images are representative illustrations of three different adrenal glands. Scale bars, 20 µm.

View larger version (69K): [in this window] [in a new window]   Figure 4. Western Blot analysis of ACs in the human adrenal cortex. Three-day cultures of human glomerulosa cells were incubated at 37 C in HBSS medium in the absence (C, Control) or in the presence of 10 nM ACTH for 5 or 15 min. Triton-insoluble fractions were prepared as described in Materials and Methods and subjected to SDS-PAGE. Proteins were analyzed by immunoblotting with specific antibodies against different AC isoforms, which were detected by chemiluminescence. For comparison purposes and loading controls, blots were stripped and reprobed with subsequent AC antibodies and actin antibody as described in Materials and Methods. Numbers and arrows indicate molecular mass of the ACs (kDa).

The measurement of cAMP production in membrane preparations can directly correlate the capacity of hormone/receptor coupling with heterotrimeric G proteins to stimulate AC activity. Morphological data, as well as Western blot analyses, indicated that human adrenal cells expressed Ca²⁺-sensitive AC1, AC3, and AC5/AC6 isoforms. AC1 and AC3 are characterized by both positive and negative calcium-regulated activity, whereas AC5/AC6 activity is decreased by Ca²⁺. To evaluate the Ca²⁺ dependence in ACTH-induced AC activation, the effect of a broad range of Ca²⁺ concentrations were compared between membranes isolated from glomerulosa or fasciculata cells, stimulated with either low (10 pM) or high (10 nM) concentrations of ACTH, which corresponds to the plateau phase of ACTH effects (6, 7). Figure 5 reveals that ACTH was able to stimulate cAMP production in the absence of calcium and that the presence of Ca²⁺ strongly affected the response to ACTH. In glomerulosa cell membrane preparations, concentrations of Ca²⁺ up to 10 nM enhanced ACTH-stimulated cAMP production. For higher concentrations, a progressive dose-dependent decrease was observed. At 100 µM of Ca²⁺, the cAMP level had dropped to the same seen in control conditions, despite the presence of ACTH (Fig. 5A). The same results were observed in fasciculata cells stimulated with 10 pM ACTH, except that the Ca²⁺ sensitivity was different at the higher concentrations of 10 nM. First, maximal increase in cAMP production was observed at 10 nM, rather than at 10 nM as seen in glomerulosa cells; and, second, high concentrations of Ca²⁺ did not abolish ACTH-induced cAMP production, which was similar to that observed in the absence of calcium (4.4-fold increase over control; Fig. 5B).

View larger version (19K): [in this window] [in a new window]   Figure 5. Effect of increasing free-Ca2+ concentrations on ACTH-induced cAMP production in membrane preparations from human adrenal zona glomerulosa (A) and zona fasciculata (B). Membranes from zona glomerulosa and fasciculata were homogenized and pelleted as described in Materials and Methods. Membranes (30 µg) were incubated with 1 mM ATP containing [32P]-ATP (106 cpm) (in a regenerating medium consisting of creatine kinase + creatine phosphate) and were incubated in buffer alone (line, basal conditions) or stimulated with 10 pM ACTH (•) or 10 nM ACTH () for 15 min at 37 C in the absence (C, Control) or in the presence of various concentrations of calcium, ranging from 10-8 M to 10--4 M. The amount of [3H]-cAMP accumulating in cells was determined and expressed as % of total intracellular [3H]-ATP and the amount of [32P]-cAMP produced in membranes as pmol of cAMP produced/min/mg protein, as described in Materials and Methods. The results are the means ± SEM of four separate experiments, each in duplicate. Basal AC activities were 2.17 ± 0.06 and 1.46 ± 0.06 pmol cAMP/min/mg of protein in human zona glomerulosa and fasciculata membrane preparations, respectively.

View larger version (36K): [in this window] [in a new window]   Figure 6. Effect of ß-subunits on cAMP production induced by ACTH in membrane preparations from human adrenal zona glomerulosa (A) and zona fasciculata (B). Thirty micrograms of membranes were incubated in buffer alone (basal conditions) or stimulated by ACTH 10 pM or ACTH 10 nM for 15 min at 37 C in the presence or absence of 100 µM QEHA peptide or 100 µM SKEE peptide in a regenerating medium consisting of creatine kinase + creatine phosphate. The amount of [3H]-cAMP accumulating in cells was determined as described in the legend to Fig. 5 and in Materials and Methods. The results are the mean ± SEM of two separate experiments, each in triplicate. *, P < 0.01, compared with ACTH-stimulated cells.

View larger version (23K): [in this window] [in a new window]   Figure 7. Effect of PTX on cAMP accumulation induced by ACTH in rat adrenal glomerulosa (A) and fasciculata (B) cells. Three-day cultured cells were labeled with 3H-adenine as described in Materials and Methods. Cells were preincubated for 18 h in the absence or presence of 100 ng/ml PTX and further stimulated with or without 10 pM or 10 nM ACTH for 15 min at 37 C in HBSS medium. Accumulated [3H]-cAMP concentrations were determined and expressed as a percentage of total [3H]-ATP, calculated as described in Materials and Methods. The results are the means ± SE of triplicate determinations in one experiment representative of two. *, P < 0.01, difference compared with ACTH-stimulated cells.

View larger version (37K): [in this window] [in a new window]   Figure 8. Effect of anti-AC antibodies on cAMP production induced by ACTH in rat membrane preparations from glomerulosa (A) and fasciculata (B) cells. Thirty micrograms of membranes were incubated in buffer alone (basal conditions) or stimulated with 10 pM or 10 nM ACTH for 15 min at 37 C in the presence of 100 ng/ml of the different AC antibodies. The amount of [3H]-cAMP accumulating in cells was determined as explained in the legend to Fig. 5 and in Materials and Methods. The results are the means ± SEM of three separate experiments, each in duplicate. *, P < 0.01, difference compared with ACTH-stimulated cells.

In the present study, we documented the cellular localization of various AC isoforms in the human adrenal gland and their sensitivity to ACTH stimulation. First, the Ca²⁺-sensitive AC1 was detected in glomerulosa and fasciculata cells. This surprising observation constitutes additional evidence that a number of neuronal markers are present in glomerulosa cells, despite their mesodermal, rather than ectodermal, origin (1). Indeed, this isoform has, so far, only been detected in the brain (25, 26). The second Ca²⁺-sensitive AC isoform, AC3, was also detected. However, although the AC1 localization is limited to the cell membrane, AC3 has both a cytoplasm and membrane localization. The other isoforms detected also have a specific subcellular localization, with AC5/AC6 and AC4 isoforms detected mainly in the cytoplasm, and AC2, as AC3, is visible in both compartments. These localizations, diffuse at the membrane level but pronounced in the cytoplasm, are compatible with the recent observations demonstrating that those isoforms—AC5/AC6, AC3, and AC4—are localized in microdomains of the plasma membranes, called caveolae, rather than in the plasma membrane itself (27, 28). Previous in situ hybridization studies have shown that the AC5/AC6 isoform is also largely expressed in heart and brain tissue (29, 30, 31) and in the rat adrenal gland (32), as seen for the ß- and PKC-sensitive AC isoforms AC2 and AC4 (for review see Refs. 29 and 31). In the human adrenal gland, the third Ca²⁺-sensitive AC isoform, AC8, as well as the third PKC-sensitive isoform, AC7, were not detected.

Some studies performed in rats have shown that stress greatly increased expression of AC2 in the hippocampus (33). In the rat adrenal gland, Shen et al. (32) have shown that ACTH treatment increased mRNA expression of AC5 and AC9, but not of AC3. Thus, physiological variations are able to modulate specific expression of AC isoforms. As has been reported in a recent publication by Lacroix et al. (34), a number of adrenal pathologies are due to the ectopic expression of AC-linked receptors. Thus, understanding the expression, biochemical properties and coupling of AC-coupled receptors (such as serotonin, GIP) would help to understand the various profiles of abnormal secretion and proliferation induced by such receptors.

Since the cloning of the nine isoforms of ACs, several studies have used cell transfection systems to understand the role, the Ca²⁺-sensitivity, and the regulation by kinases of the various AC isoforms (35). From these studies, it is now clearly established that AC1 and AC3 have a biphasic response to Ca²⁺ and are inhibited by the heterotrimeric Gi protein (both via i- and ß-subunits); that AC5/AC6 is inhibited by Ca²⁺ and PKA; and that AC2, AC4, and AC7 are stimulated by ß and PKC (10, 11, 12). We have, thus, attempted to verify some of these properties in the human adrenocortical cells stimulated with ACTH. The results shown in Fig. 5 are compatible with the reported biphasic action of Ca²⁺ on AC1 and AC3 activity described in the literature, as well as in GnRH neurons (36), and are in agreement with our previous observations that, in human adrenocortical cells, cAMP accumulation is under positive feedback regulation controlled not only by PKA, but also by Ca²⁺ (7). However, results in Fig. 8, obtained with the optimal concentration of calcium (10 nM), also clearly indicate that AC1 and AC3 are not sensitive to ACTH, at least in the zona glomerulosa, and that only AC3 is modulated by ACTH in the zona fasciculata. These latter observations support several studies indicating that, both in vivo and in vitro, calcium channel blockers have only a moderate inhibitory action on ACTH-induced aldosterone and cortisol secretion (37, 38, 39), thus suggesting that calcium modulates, rather than being essential to primary AC activation. Calcium ions required for the modulation of the calcium-sensitive AC isoforms may originate from various sources. We have previously shown that ACTH modulates calcium, potassium, and even chloride channels (15, 40, 41). Moreover, voltage-independent channels of the TRP family, such as TRP4, detected in bovine adrenal cortex (42) and rat (M. D. Payet, unpublished observations) may also be involved.

A very interesting novel observation is that ACTH modulates ß-sensitive AC isoforms. Indeed, the AC2 and AC4 isoforms, in addition to Gs stimulation, are positively modulated by the ß subunits of G proteins and by PKC (43, 44, 45, 46). As shown in Fig. 6, the addition of a ß sequestrant peptide, QEHA, decreased ACTH-stimulated cAMP production, further supporting the observation that the ACTH receptor is tightly coupled to AC2 and/or AC4 via ß-subunits. Moreover, treatment of cells with PTX, which inactivates i, enhanced ACTH-induced cAMP production both in the zona glomerulosa and in the zona fasciculata. Thus, the net effect of G_i activation is a stimulation of cAMP production via the ß-subunits and not an inhibition through i. This observation indicates that ACTH is mainly coupled to the ß-sensitive AC2/AC4 and not (zona glomerulosa) or less (zona fasciculata) to the i-sensitive AC3, corroborating results observed in Fig. 8. These results reinforces the observation that ACTH may have a number of actions mediated by the ß-subunits of G proteins, as shown recently in rat adrenal gland in the modulation of chloride channels (15) or in the increase in p42/p44^MAPK activity in the Y1 adrenocortical cell line (47).

Altogether, two important conclusions can be raised from this study. First, the human adrenal gland expresses several isoforms of AC, each with different cellular and subcellular localizations, and, second, that ACTH works primarily through AC5/AC6, AC2, and AC4, whereas the activation of AC1 and AC3 may occur consequently to calcium influx, at least in the zona glomerulosa. Compilation of the present data, together with our previous studies and the biochemical properties of AC isoforms described in the literature, we propose a model that could explain why the ACTH response is sustained rather than transient and why the amplitude of the response to ACTH is so high compared with many other stimuli. Indeed, even if all the cyclases were activated by the G protein s-subunit the overall cAMP production and accumulation could be modulated by the specific temporal regulation by calcium, the ß-subunits of G proteins or by other second messengers. We suggest that ACTH first stimulates the AC5/AC6 isoform (to produce cAMP in the absence of calcium (7, 8, 13) (Fig. 9A, step 1). Then, via cAMP and PKA, ACTH activates a slow but sustained Ca²⁺ influx through L-type channels (Fig. 9A, step 2) (7, 40, 41), hence activating the AC3 isoform (Fig. 9B, step 1), at least in fasciculata cells. Once Ca²⁺ rises to a critical value, inhibition of AC5/AC6, as well as AC3, occurs (Fig. 9B, steps 2 and 3; also Fig. 5); PKA also inhibits AC5/AC6 (Fig. 9A, step 3) (10, 12). Moreover, PTX treatment indicates that ACTH is linked not only to a G_s, but also to a G_i protein (Fig. 9C, step 1) (48). Activation of i contributes to the inactivation of AC3 and AC5/AC6 (Fig. 9C, steps 1 and 2) (see also Fig. 7), although the release of the ß-subunits could activate AC2 (see Fig. 6), the only isoform not sensitive to Ca²⁺ (Fig. 9C, step 3). This sequence illustrates why ACTH is such a potent stimulus of cAMP production in the adrenal gland.

View larger version (44K): [in this window] [in a new window]   Figure 9. Model for ACTH-induced cAMP production in adrenocortical fasciculata cells. According to the present results and those described previously, ACTH could stimulate AC5/AC6 isoforms (A, step 1), which, through cAMP and PKA, activate a slow but sustained Ca2+ influx through L-type channels (A, step 2). At the same time, PKA inhibits AC5/AC6 (A, step 3) and Ca2+ activates AC3 isoform (B, step 1). When Ca2+ rises to a critical value, it inhibits AC5/AC6 and also AC3 (B, step 2 and 3); moreover, PTX treatment indicates that ACTH is not only linked Gs, but also to Gi protein. Activation of i contributes to the inactivation of AC3 (C, steps 1 and 2), although release of ß could activate AC2 (C, step 3), the only isoform that is not sensitive to Ca2+. Meanwhile, ß-subunits may stimulate other effectors such as phospholipase Cß3 isoform, MAPK cascade, or cationic channels.

Acknowledgments

We thank Quebec Transplant for the gift of human adrenal glands, Lucie Chouinard for experimental assistance, Dr. Jacques Hanoune (INSERM U99, Creteil, France) for very stimulating discussions, and Dr. Christophe Breton (Laboratoire d’Endocrinologie des Annélides, UPRESA CNRS8017, Université des Sciences et Technologies de Lille, Lille, France) for both stimulating discussions and invaluable technical assistance and collaboration. We are also indebted to Dr. Nuria Basora for critical review of our manuscript.

Footnotes

This work was supported by grants from the Medical Research Council of Canada (to M.D.P. and N.G.-P.).

Abbreviations: AC, Adenylyl cyclase; PTX, pertussis toxin; TBS, Tris-buffered saline.

Received March 5, 2001.

Accepted May 9, 2001.

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