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Service of Endocrinology (M.C., N.G.-P.), Department of Medicine, Department of Physiology and Biophysics (M.D.P., E.R., N.G.-P.), Faculty of Medicine, University of Sherbrooke, Sherbrooke (Québec) Canada J1H 5N4; INSERM U469 (G.G.), 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, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Québec, Canada J1H 5N4. E-mail: ngallo01{at}courrier.usherb.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The intracellular concentration of cAMP is modulated, in part, by the activities of one or more adenylyl cyclase (AC) isoforms (4) and one or several cyclic nucleotide phosphodiesterases (PDEs) (5, 6). To date, little is known as to the regulation of intracellular cAMP accumulation under ACTH stimulation. Indeed, in most studies, cAMP was measured in conditions where PDE activity was blocked by addition of a nonselective PDE inhibitor, IBMX (3-isobutyl-1-methyl-xanthine) or theophilline. Nine different PDE gene families and more than 40 different isozymes are now described (6, 7, 8, 9). Four isozyme families are known to efficiently hydrolyze cAMP at physiological concentrations of substrate, namely: 1) Ca2+-calmodulin-regulated PDEs (CaM-PDEs called PDE1), which hydrolyze both cAMP and cGMP; 2) cGMP-stimulated PDEs (cGS-PDEs called PDE2), which hydrolyze cAMP; 3) cGMP-inhibited PDEs (cGI-PDEs called PDE3), which have a high affinity for cAMP and are inhibited by low concentrations of cGMP; and finally, 4) cAMP-specific PDEs (PDE4), which possess high selectivity for cAMP (5, 9, 10, 11). How these different PDEs contribute to the regulation of cAMP levels induced by ACTH remains to be determined.
The aim of this study, therefore, was to characterize the role of the different subtypes of PDEs in the control of basal and ACTH-stimulated cAMP production. We compared the effect of selective inhibitors of cyclic nucleotide PDEs [EHNA (erythro-9-[2-hydroxy-3-nonyl]adenine) for PDE2, LY195115 for PDE3, and rolipram for PDE4] with the nonselective inhibitor, IBMX, on cAMP production by AC activation, PDE activation, and cAMP accumulation in glomerulosa cells. These studies were performed both in rat and human cells, because previous studies from our laboratory indicated important differences in ACTH-stimulated cAMP accumulation between these two models (1, 12).
We clearly demonstrate that ACTH controls cAMP accumulation via multiple mechanisms: a rapid and sustained activation of AC, a rapid inhibition of PDE2 activity, and a delayed activation of PDE2. Moreover, this involvement is more important in rat than in human glomerulosa cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[32P]-ATP (3000 Ci/mmol), [2,8-3H]-cAMP
(24 Ci/mmol), Biotrak cGMP enzyme immunoassay (EIA) from Amersham Pharmacia Biotech (Oakville, Ontario, Canada); ATP, cAMP, GTP,
cGMP, adenosine, guanosine, EDTA, dithiothreitol, 5'-nucleotidase
crotalus venom, EHNA, and deoxyribonuclease from Sigma Chemical Co. (St. Louis, MO); ACTH 1–24 peptide (Cortrosyn) from
Organon Canada (Toronto, Canada); aldosterone antiserum
from ICN Biochemicals, Inc. (Costa Mesa, CA);
[3H]-aldosterone (72 Ci/mmol) from NEN Life Science Products (Boston, MA); creatine kinase, creatine phosphate
disodium and EGTA from Roche Molecular Biochemicals
(Montréal, Canada); collagenase, MEM, and OPTI-MEM medium from
Life Sciences, Inc. (Burlington, Ontario, Canada);
rolipram (Schering AG),
1,3-dihydro-3,3-dimethyl-1-methyl-5(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)2H-indol-2-one
(LY195115) (Eli Lilly & Co.). All other chemicals
were of A-grade purity.
Preparation of glomerulosa cells
Human adrenal glands were obtained from renal transplant donors,
16–22 yr old, through a collaboration with the
Quebec-Transplant Association. This project was approved by the
Human Subject Review Committee of our institution. After removal,
glands were kept on ice in McCoy’s medium and transported within
4 h to the laboratory. Glands were processed as previously
described (1, 13). Briefly, adrenal glands were cleansed of fat, cut
into small flat sections, then further into thin slices. Capsule and
zona glomerulosa (first slice) were used to prepare glomerulosa cells.
Rat glomerulosa were obtained from adrenal glands of female Long Evans
rats, weighing 200–250 g, and were isolated according to the method
described in detail elsewhere (14). Briefly, the successive steps of
zona glomerulosa isolation and cell dissociation were performed in MEM
Eagle medium (supplemented with 100 U/ml penicillin and 100 µg/ml
streptomycin). After a 20-min incubation at 37 C in collagenase (2
mg/ml, 4 capsules/ml) and deoxyribonuclease (25 µg/ml), the cells
were disrupted by gentle aspiration with a sterile 10-ml pipette,
filtered, and centrifuged for 10 min at 100 x g. They
were then resuspended in OPTI-MEM medium supplemented with 2% FBS, 100
U/ml penicillin, and 100 µg/ml streptomycin and plated in 35-mm
tissue culture dishes at a density of approximately 1 x
105 cells/dish. The cells were cultured at 37 C in a
humidified atmosphere of 95% air-5% CO2. The culture
medium was changed every day, and the cells were used after 3 days of
culture. At this time, cell density was approximately 3.0 x
105 cells/dish.
Membrane preparation
The zonae glomerulosa were 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 were centrifuged at
150 x g for 10 min. The supernatant was then
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. On the day of the experiment, membranes
were washed three times in buffer containing 50 mM Tris-HCl
(pH 7.6), 2 mM EGTA, and 1 mM DTT.
cAMP determination
Intracellular cAMP production was determined by measuring the
conversion of [3H]-ATP into [3H]-cAMP, as
previously described (3). In short, cultured cells were incubated at 37
C in OPTI-MEM culture medium containing 2 µCi/ml
[3H]-adenine. After 1 h, the cells were washed with
cold Hanks’ buffered saline (HBS: NaCl, 130 mM;
KCl, 3.5 mM; CaCl2, 1.8 mM;
MgCl2, 0.5 mM; NaHCO3, 2.5
mM; HEPES, 5 mM) supplemented with 1 g/L
glucose and 0.1% BSA. Cells were incubated in the same buffer
containing 1 mM IBMX or 25 µM of selective
PDE inhibitors for 15 min at 37 C. ACTH
(10-11–10-7 M) was then added to
the incubation medium for an additional 15 min at 37 C. The reaction
was ended by aspiration of the media and addition of TCA 5%. Cells
were scraped with a rubber policeman, and 100 µl of a cold solution
of ATP and cAMP (5 mM each) was added to the mixture.
Cellular membranes were pelleted at 5,000 x g for 15
min, and the supernatants were sequentially chromatographed on Dowex
and Alumina columns, according to the method of Salomon (15), allowing
the separation of [3H]-ATP nucleotide (primarily
[3H]-adenine) from [3H]-cAMP. cAMP
formation was expressed as: percent conversion =
[[3H]-cAMP/([3H]-cAMP +
[3H]-ATP)] x 100 per 15 min by 106 cells. It
should be noted that EHNA is not only a PDE2 inhibitor but also an
adenosine deaminase inhibitor. Indeed, addition of EHNA decreased the
quantity of adenosine incorporated in the ATP pool by 30%. However,
this decrease did not affect our results because ATP is in excess,
compared with cAMP produced (around 0.05%); furthermore, our results
are expressed as a ratio of stimulation between stimulated over control
cells.
AC activity
Membranes were incubated for 10 min at 37 C in 60 µl
containing 50 mM Tris-HCl (pH 7.6), 5 mM
MgCl2, 1 mM ATP (containing
[32P]-ATP 106 cpm), 0.25 mg/ml creatine
kinase, 1.3 mg/ml creatine phosphate, and [3H]-cAMP
[20,000 cpm, with or without ACTH (10 nM)], IBMX (1
mM), or PDE inhibitors (25 µM), as described
by Méry et al. (16). The reaction was initiated by the
addition of membranes (30 µg/assay) and halted with the addition of
500 mM HCl. The amount of [32P]-cAMP
formed was separated on Alumina columns, and resulting activity was
expressed as pmol of cAMP produced/min/mg protein, after correction for
the recovery of [3H]-cAMP.
PDE activity
The membranes were incubated, with or without ACTH (10
nM), in the presence or absence of PDE inhibitors (25
µM), in 40 mM Tris (pH 7.5), 2 mM
MgAc2, 0.01 mM CaCl2, and 1 mg/ml
BSA, as described by Méry et al. (16), using the
two-step assay procedure described by Thompson et al. (17).
Reactions were initiated by addition of an appropriate amount of
membrane (30 µg/assay), yielding approximately 15% substrate
hydrolysis. Incubation was performed at 37 C with addition of cAMP
(10-6 M) + [3H] cAMP (20,000
cpm) for 10 min. The reaction was stopped with the addition of IBMX (1
mM), cAMP (10 mM), and cGMP (10
mM), followed by incubation with snake venom (containing
5'-nucleotidase activity) for 15 min at 37 C. The crotalus
atrox venom used in these studies exhibited no appreciable PDE
activity. The snake venom reaction was stopped with addition of
guanosine (0.1 mM), adenosine (0.1 mM), and
EDTA (0.015 M). The [3H]-adenosine formed was
separated on QAE Sephadex A25 columns (Sephadex Anion Exchange). PDE
activity is expressed as pmol of cAMP hydrolyzed/min/mg protein.
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B). However, the half-maximal
effective concentration (EC50, 1 nM), threshold
(0.1 nM), and plateau (100 nM) were the same in
both models. In human cells, omission of IBMX from the incubation
medium decreased the effect of ACTH concentrations up to 0.1
nM by 22% but not the lower concentrations (Fig. 1A). In
contrast, in rat cells, a decrease was observed for all concentrations
used, with a maximal inhibitory effect of 47% at 100 nM
(Fig. 1B). Moreover, when cAMP production was measured as AC activity
in membrane preparation, in basic medium (without cGMP and IBMX), the
stimulatory effect of ACTH was similar in human (2.88 ± 0.59-fold
increase, n = 3, P < 0.001) and in rat membrane
preparations (2.58 ± 0.48-fold increase, at the concentration of
100 nM, n = 3, P < 0.01) but lower
than that observed in human and rat cells (11 ± 1 and 24 ±
2-fold, respectively). Addition of 1 mM IBMX did not
significantly affect basal value but slightly increased the
ACTH-stimulated level of cAMP (Fig. 1, C and D). It should be noted,
moreover, that the basal level of cAMP production is higher in human
(2.5 ± 0.2 pmol/min/mg) than in rat membranes (0.67 ± 0.12
pmol/min/mg).
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, circle). In rat cells, the
significant increase in cAMP was delayed up to 5 min (12.3 ± 1.5
fold increase), reached a maximal level (29.4 ± 2.5-fold
increase, n = 3) after 15 min, then decreased (Fig. 2B, circle). We also studied the effect of PDE inhibitors on the
time-course effect of ACTH. In human cells, addition of 25
µM LY195115 (PDE3 inhibitor) or 25 µM
rolipram (PDE4 inhibitor), together with ACTH, induced a time-dependent
increase in cAMP (triangle). During the first 20 min,
stimulation was slightly lower than that with ACTH alone but reached
similar levels (13.4 ± 0.9 and 15.3 ± 1.2-fold increase,
respectively) to those observed during the plateau phase of ACTH action
(Fig. 2A). In contrast, in rat cells, 25 µM rolipram or
25 µM LY195115 did not affect the kinetics of
ACTH-stimulated cAMP levels (Fig. 2B, triangle). In similar
experimental conditions, 25 µM EHNA strongly enhanced
cAMP production induced by ACTH (square) in rat and human
cells. The effect was evident as early as 1 min after stimulation and
remained elevated up to 40 min (Fig. 2, A and B). All these results
indicate a role for PDE2 in the regulation of cAMP accumulation, which
was higher in rat than in human cells.
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). As expected, 10 nM
ACTH (filled circle) induced a time-dependent increase in
cAMP production, with a maximal effect at 15 min (3.8 ± 0.3-fold
increase, n = 3), which decreased to control levels after 15 min
of incubation (IBMX was omitted in these experiments). Addition of 10
µM cGMP (diamond) blocked cAMP production
induced by ACTH, whereas a preincubation with 25 µM EHNA
(square) accelerated cAMP production, compared with ACTH
alone, with a maximal effect at 5 min (3.9 ± 0.4-fold increase,
n = 3), followed by a small decrease. These results confirmed the
role of PDE2 in the increase in ACTH-induced cAMP production.
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), the effect of ACTH on PDE activity was only
observed after a 10-min incubation and was maintained for at least 25
min (Fig. 4, filled circle),
with a maximal stimulation of 5.0 ± 0.2-fold increase, n =
3, P < 0.001. When membranes were incubated with 10
µM cGMP (diamond), stimulation of PDE activity
was time dependent, with a 1.7 ± 0.3-fold increase as early as 1
min, with a plateau obtained after 20 min (7.6 ± 0.3-fold
increase). Preincubation with 25 µM EHNA
(square) inhibited PDE activity, indicating its PDE2 nature.
The effect was dose dependent in human and rat membrane preparations,
with an EC50 of 10 pM and 0.1 nM,
respectively (Fig. 5, A and B), but not
in the cytosolic fraction (data not shown). The effect was higher in
rat (4.2 ± 0.4-fold increase, n = 3, P <
0.001) than in human (2.2 ± 0.5-fold increase, n = 3,
P < 0.01) at a concentration of 100
nM.
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) and rat (Fig. 6B)
membranes. Addition of cGMP increased PDE activity in a dose-dependent
manner, with a strong inhibition at 10 µM. A 10-min
preincubation with 25 µM EHNA, before addition of cGMP,
blocked the stimulatory effect of cGMP, indicating that the increase in
PDE activity by cGMP was caused by activation of PDE2.
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)
and rat (Fig. 7B) cells. This drop in cGMP occurred rapidly (within 5
min of stimulation) and reached maximal effect after 10 min. The effect
was also dose-dependent and was observed even at low concentrations of
ACTH (EC50 = 1 pM). Maximal inhibition was
observed at 1 nM (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dose-response curve analyses show that cAMP production in glomerulosa cells from adult human and rat adrenal are very sensitive to ACTH. However, dose-response and time-course comparative studies between cells and membranes indicate that the intracellular level of cAMP differs in the two models and that a cGS-PDE2 is involved in these effects. Using selective pharmacological inhibitors such as EHNA, originally designed as an inhibitor of adenosine deaminase (20), but recently shown to be a selective inhibitor of myocardical PDE2 at concentrations in the micromolar range (21), we were able to demonstrate that PDE2 controls the level of intracellular cAMP induced by ACTH and that PDE2 itself is under ACTH control. After a 10-min preincubation of cells with selective PDE inhibitors, EHNA increased the accumulation of cAMP induced by ACTH to a level close to that seen with IBMX, a nonselective inhibitor of cyclic nucleotide PDEs.
The basal level of cAMP production was higher in human than in rat, although the stimulating effect of ACTH was 2-fold lower in human than in rat membrane preparations. Moreover, maximal stimulation was 11-fold higher in cells vs. membranes. These results suggest either a differential expression of the AC isoforms in the two models or differential regulation by cyclic nucleotide PDEs. Either hypothesis may be true. Indeed, recent studies from Shen et al. (22) indicate that AC5 and AC6 are expressed in rat glomerulosa cells, whereas Burnay et al. (23) showed that AC3 is expressed in bovine adrenal cells. Our results (data not shown) also indicate that AC 6, AC7, and AC3 are the isoforms present in human glomerulosa cells. Moreover, time-course studies indicate different involvement of PDE2 in the two models. The early phase of ACTH action was enhanced by PDE2 inhibition in rat cells, although the decrease observed after a 15-min incubation suggests PDE2 activation at that time. cAMP stimulation in human cells was sensitive to PDE2 inhibition immediately upon addition of ACTH. Incubation with EHNA further confirms that PDE2 is strongly involved in the regulation of cAMP level. Its effect occurred rapidly (within 1 min of incubation) and induced an increase in cAMP accumulation up to 40 min, strongly modifying the kinetics of cAMP hydrolysis. By comparison with the effect produced by EHNA, other PDE inhibitors such as LY195115 [cGI-PDE inhibitor, (5)] and rolipram [cAMP-specific PDE inhibitor (19)] had no effect, suggesting that PDE3 and PDE4 are poorly (or not at all) involved in the regulation of intracellular cAMP induced by ACTH. These results also indicate that the increase in cAMP accumulation produced by ACTH is caused not only by AC activation but also by a biphasic effect of ACTH on PDE2 activity, e.g. an initial inhibition caused by an decrease of cGMP levels by ACTH, followed by a stimulation, caused by cAMP itself.
Indeed, a direct effect of ACTH on PDE activity was demonstrated, although only after 10 min. The PDE2 nature of the ACTH-sensitive PDE activity was confirmed by two results. First, EHNA completely blocked ACTH-induced PDE2 activity; and second, cGMP alone controlled PDE activity. At submicromolar concentrations, cGMP binds the regulatory site of PDE2, causing a 3- to 10-fold activation of cAMP hydrolysis. At concentrations up to 50 µM, cGMP binds the catalytic site as well, thus inhibiting its action (24, 25). Moreover, it is known that cGS-PDEs and most CaM-PDEs exhibit a higher affinity for cGMP than cAMP, whereas cGI-PDEs display high affinity for both cGMP and cAMP.
Previous studies have shown that cGMP-stimulated PDE2 is the predominant isoform of PDE present in the adrenal cortex (26), with its highest concentration found in the zona glomerulosa (27), at least in bovines. This cGMP-stimulated PDE is involved in the inhibitory effect of atrial natriuretic peptide (ANP). Indeed ANP decreases cAMP and steroidogenesis through the activation of PDE2 (27). Positive hormonal regulation of PDE has been described in several cell types (5). For example, it has been shown that agents or hormones that increase cAMP also produce a rapid increase in the cGMP-inhibited PDE3, such as glucagon in rat liver or isoproterenol in platelets (28). By contrast, inhibition of membrane-bound PDE by hormones is not a commonly observed occurrence, but it has been previously described for glucagon in frog and mouse cardiac ventricle. In this particular example, glucagon increased cAMP only via inhibition of a cGI-PDE, without any effect on AC activation (16, 29).
In agreement with the effect of ACTH on PDE activity, which is cAMP-specific, it is not surprising that, in our conditions, ACTH induced a rapid and strong time-dependent decrease in cGMP content, corroborating previous studies of Elliot and Goodfriend (30) and Matsuoka et al. (31) in bovine zona glomerulosa. These results differ from those of Nambi et al. (32), in bovine zona fasciculata, where an increase in cGMP was observed over a low range of concentrations, from 0.1–7 pM. For higher concentrations (up to 100 pM), cGMP levels returned to basal values, and even below (32), whereas cAMP production was stimulated in a dose-dependent manner. These differences may be caused by cell-specific properties. Indeed, in rat and human glomerulosa cells, stimulation with S-nitroso-N-acetylpenicilamine, an exogenous source of NO, also failed to stimulate cytosolic cGMP production (33). A further argument to support these cell-specific properties is also demonstrated by the action of ANP, which stimulated cGMP in glomerulosa cells but not in fasciculata cells (34). Finally, these differences may be caused also by a higher expression of PDE2 in the zona glomerulosa than in the zona fasciculata (26).
As previously suggested by Hamet et al. (35), our results
indicate that, in glomerulosa cells (and, in particular, human
glomerulosa cells, where the basal content of cGMP is very high), basal
PDE2 activity may be sufficient to hydrolyze the primary burst of cAMP
induced by ACTH. In accordance with this hypothesis is the observation
that AC activation in membranes is stimulated at lower concentrations
of ACTH (a 2-fold increase at 0.01 nM), whereas the initial
increase in cells is observed only at 0.1 nM (see Fig. 1).
Moreover, the threshold of cGMP inhibition is lower (0.1
pM) than the threshold of AC activation (10
pM), further indicating that a larger increase in cAMP is
prevented by basal PDE2 activity, whereas the plateau in cAMP
production is attributable to the inhibition of PDE2 subsequent to a
decrease in cGMP by ACTH.
In spite of the studies of Sharma et al. (32, 36, 37, 38), the role of cGMP in steroid secretion has been described as nonessential (30). Our data reconcile these apparent discrepancies and indicate that cGMP may be more important in the regulation of PDE2 activity (and thus, cAMP production), rather than in steroidogenesis directly. However, how ACTH decreases cGMP remains to be determined. It may be via a direct action of ACTH on inhibition of a nitric oxide synthase, this pathway being present in glomerulosa cells (33). Alternatively, its effect could be mediated by cGMP-specific PDE6.
These studies, together with those described previously, indicate that ACTH action is complex. In the present study, we demonstrate that the increase in cAMP production induced by ACTH is the result of a balance between activation of AC and direct modulation of a PDE2 activity, an effect mediated by inhibition of cGMP content. Moreover, we have also previously shown that the Ca2+ ion is essential for cAMP accumulation and aldosterone secretion (1). Several studies, including ours, indicate that Ca2+ action on ACTH-induced secretion is provided through Ca2+ influx (39, 40, 41). Regulation of Ca2+ influx is itself controlled by ACTH action on T- and L-type Ca2+ channels (42, 43, 44) but also on K+ channels (45) and even Cl- channels (46). However, the exact primary action of Ca2+ is not yet clearly established. For example, in the rat, the AC isoforms detected are not Ca2+-sensitive (22), whereas in human cells, Western blot analyses indicate the presence of at least AC3, which is Ca2+-sensitive (unpublished results), indicating again a differential regulation in both models. Our recent work indicates that the cytoskeleton may be the primary target of Ca2+-ACTH action. Indeed, ACTH induces a rapid translocation of actin fibers from the cytosol to the membrane, a relocalization that is blunted in a Ca2+-free medium (47). All these observations are consistent with previous classical studies (39, 48, 49) indicating the important role of Ca2+ in mediating ACTH response. However, in spite of this progress, one important question remains: Is the differential involvement of Ca2+, phospholipase C (3, 40, 50), cAMP, and PDE (observed at low and high concentrations of ACTH) caused by ACTH binding first to a high-affinity site and then to a low-affinity site (51, 52, 53, 54); or is this involvement caused by differential binding of N-terminal and C-terminal ACTH molecules at different sites on the ACTH receptor, each mediating its own signal transduction pathway (55, 56, 57)?
In conclusion, this study is the first to describe that PDE2 activity controls the production of cAMP induced by ACTH, an effect mediated by a direct action of ACTH on PDE2 activity. In a first step, ACTH inhibits PDE2 activity, by inhibiting cGMP production. In a second step, once cAMP reaches its plateau phase, cAMP itself stimulates PDE2 to decrease cAMP to a basal state of production. Taken together, these results indicate that ACTH stimulation controls PDE2 activity, which together with Ca2+ and the cytoskeleton, participates in the regulation of cAMP accumulation in glomerulosa cells; the role of each participant having similarities, but also differences, in the human and rat models.
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Acknowledgments |
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Footnotes |
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Received December 2, 1998.
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
) or with (•) the nonselective PDE inhibitor, IBMX (1
mM), before addition of increasing concentrations of ACTH,
for a subsequent incubation period of 15 min. The amount of
[3H]-cAMP accumulating in cells was determined and
expressed as percent of total intracellular [3H]-ATP, and
the amount of 
),
LY195115 (25 µM, 
), and Rolipram (25 µM,
), and then further incubated with 10 nM ACTH for
various time periods. The amount of [3H]-cAMP
accumulating in cells was determined and expressed as percent of total
intracellular [3H]-ATP, as described in Materials
and Methods. Results for rat glomerulosa cells are the
mean ± SEM of three experiments, each in triplicate.
Results for human glomerulosa cells are the mean ±
SEM of one experiment, in triplicate.
), and then further incubated without (
) or with
ACTH, after a 10-min preincubation with 25 µM EHNA (
) a 10-min preincubation at 37 C
with the specific cGMP-stimulated-PDEs inhibitor, EHNA (25
µM). PDE activity was measured by a double-step
radioenzymatic assay involving [3H]-cAMP and snake venom
reaction (containing 5'-nucleotidase activity), as described in
Materials and Methods. Results are the mean ±
SEM of three experiments, each in duplicate.
