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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 (Gs) is coupled to activation of AC, whereas an
inhibitory G protein (Gi) 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).
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).
Corroborating morphological data, AC1 was detected in the
Triton-insoluble fraction and the level of detection did not change
during ACTH stimulation. The AC3 level increased after 15 min of ACTH
stimulation, suggesting that ACTH increased the association of AC3 with
the components of the Triton-insoluble fraction (membranes or
cytoskeleton), as suggested previously (22, 23). In
contrast, AC2 was detected in the Triton-insoluble fraction in basal
conditions but decreased after ACTH-stimulation. For the AC5/AC6
isoforms, the overall amount localized in the Triton-insoluble fraction
did not vary in the presence of ACTH.
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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).
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-subunits are involved in AC activation,
membranes were incubated with the QEHA peptide. This peptide encodes
amino acid residues 956–982 of the C2a region of AC2, which interact
with Gß via a scavenging effect, thereby preventing activation of
ß-sensitive AC2 (16). Incubation with 100
µM QEHA peptide (15, 16, 24) did not modify
basal AC activity but significantly decreased the cAMP production
induced by 10 pM and 10 nM ACTH in both
glomerulosa and fasciculata membranes (Fig. 6). As a control, the effect of SKEE
peptide, which encodes the same corresponding region of the AC3
isoform, but which is not ß sensitive, had no effect on ACTH
stimulation, in either glomerulosa or fasciculata membrane preparations
(Fig. 6).
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-subunits involved in
AC activation, or various cellular responses, are usually released
following Gi activation. Cells were preincubated for 18 h in the
presence of PTX (100 ng/ml) and were then stimulated with ACTH. As
shown in Fig. 7, A and B, PTX did not
significantly modify the effect of low concentrations of ACTH on cAMP
production, but strongly increased cAMP levels induced by the higher
concentration of ACTH. These results indicate that, in addition to Gs
coupling, ACTH receptor is also coupled to a Gi protein, probably
through two opposite regulatory processes, mediated by i- (for
inhibition) and ß-subunits (for activation).
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, incubation with
either the anti-AC2 or the anti-AC5/AC6 antibodies decreased
ACTH-induced cAMP in both cell types and at both concentrations of ACTH
used. Incubation with the anti-AC1 antibody had no effect on ACTH
action on either glomerulosa or fasciculata membrane preparations
whereas anti-AC3 antibody decreased cAMP levels only in ACTH-stimulated
fasciculata membranes. These results indicate that human adrenocortical
cells are more sensitive to the Ca2+-inhibitable
AC5/AC6 isoform and to ß-sensitive ACs, than to
Ca2+-stimulated isoforms.
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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
Gi 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/p44MAPK 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 Gs, but also to a
Gi 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.
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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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) 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 
