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Faculty of Integrated Arts and Sciences (T.Y., K.H., S.K.), Hiroshima University, Higashihiroshima 739, Japan; Department of Biophysics and Life Sciences (T.K., Y.O., S.K.), Graduate School of Arts and Sciences, University of Tokyo at Komaba, Meguro, Tokyo 153, Japan
Address all correspondence and requests for reprints to: Takeshi Yamazaki or Shiro Kominami, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashihiroshima 739, Japan. E-mail: takey{at}ipc.hiroshima-u.ac.jp
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Calcium ion, Ca2+, has been suggested to be a second messenger for ACTH action in adrenal fasciculata-reticularis cells because steroidogenesis of the cultured cells was stimulated by addition of Ca2+ in the medium both in the presence and absence of ACTH (6). Extracellular Ca2+ is required for binding of ACTH to its receptor and also for maximum stimulation of steroidogenesis (7). At low ACTH concentrations, Ca2+ influx via T-type Ca2+ channels might participate in the stimulation of steroidogenesis in bovine fasciculata cells (8, 9). Increase of cellular Ca2+, however, is barely detectable in ACTH-stimulated cells with conventional methods (10, 11). Recently, we have observed Ca2+ signals induced by 0.1–100 pM ACTH in individual cultured bovine adrenal fasciculata cells using a microscopic fluorescence measurement technique (12). This observation is strong evidence for a role for Ca2+ as a second messenger of ACTH action.
Physiological second messenger(s) for ACTH action had been discussed for a long time. Application in the pM range of ACTH stimulated steroidogenesis in fasciculata-reticularis cells without significant increase in cellular cAMP synthesis (3, 13, 14). We have reported that 15-lipoxygenase metabolite(s) of arachidonic acid is a second messenger for ACTH at the physiological condition because pM concentrations of ACTH-stimulated bovine adrenal 15-lipoxygenase activity, which corresponded well with the stimulation of the steroidogenesis and addition of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a 15-lipoxygenase metabolite of arachidonic acid, stimulated steroidogenic activity of the cells (14). The action of ACTH in bovine fasciculata-reticularis cells may be mediated by at least three messengers: cAMP, Ca2+, and the lipoxygenase metabolite(s). On the other hand, the second messenger system of NPS-ACTH is expected to be simpler because cAMP likely does not have a significant role in the action of the modified peptide.
In this report, we clarified the roles of Ca2+ and lipoxygenase metabolite(s) as second messengers for the acute action of NPS-ACTH on the steroidogenesis in cultured bovine adrenal fasciculata-reticularis cells. The relative importance of second messengers, i.e. cAMP, Ca2+ and lipoxygenase metabolite(s), in the acute stimulation by physiological concentrations of ACTH was also characterized.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture, steroidogenic activity, and cAMP analysis
Fresh adrenal glands of Holstein-Friesian cows were obtained
from a local slaughterhouse, and fasciculata-reticularis cells were
isolated as described (17). Contamination of glomerulosa cells was less
than 2% for each preparation because no aldosterone synthesis was
detected in culture medium by specific RIA after incubation of the
cells without addition of any inhibitors (18). Cells grown to
confluence on a 24-well culture plate (Sumitomo Bakelite, Tokyo, Japan)
were incubated with a fresh medium containing the pregnenolone
metabolism inhibitors, 2 µM trilostane and 20
µM SU-10603 (kindly supplied by C. R. Jefcoate), and
steroidogenic stimulators, i.e. ACTH, NPS-ACTH, or dibutyryl
cAMP ((Bu)2cAMP, Boehringer Mannheim,
Mannheim, Germany). Various inhibitors, i.e. 40
µM nicardipine (Sigma Chemical Co., St.
Louis, MO), and 50 µM AA-861 (Wako, Osaka, Japan)
were added 15 min before beginning the incubation for metabolism of
cholesterol to pregnenolone. After 2 h incubation of the cells
under 5% CO2 at 38.5 C, accumulated pregnenolone in the
medium was extracted with hexane and measured by specific RIA, as
described previously (19). Cell morphology did not change during the
2 h incubation with the inhibitors.
For measurement of cAMP accumulation, the cells were incubated for 2 h with fresh medium containing 0.2 mM 3-isobutyl-1-methylxanthine (Sigma Chemical Co.). The cAMP accumulated in the cells was extracted with ethanol and analyzed with a Biotrak cAMP EIA system (Amersham International, Amersham, UK) as described (14).
Microscopic imaging of Ca2+ fluorescence and its
analysis
For Ca2+ imaging, cells were grown in glass-bottom
dishes coated with collagen. The cells were loaded for 1 min at 37 C
with 5 µM Fura-2/AM (Dojin, Kumamoto, Japan) in the
presence of 0.01% Triton X-100 in 1 ml of physiological salt solution
(PSS) containing 120 mM NaCl, 4 mM KCl, 1.25
mM CaCl2, 1 mM NaHPO4,
0.5 mM MgSO4, 5 mM
NaHCO3, 0.1% BSA, 0.1% glucose, and 10 mM
HEPES (pH 7.4). After three washes with PSS, the cells were
preincubated for 20 min with or without 40 µM nicardipine
at 37 C. The glass-bottom dish was mounted on a video-enhanced
fluorescence microscope as previously described (12). The cells were
excited by 340 and 380 nm in the presence of various concentrations of
NPS-ACTH, and fluorescence intensity was recorded on video tape. After
digitizing the intensity of the recorded fluorescence,
Ca2+ concentrations in individual cells
were estimated from image analysis of fluorescence intensity ratio
between excitation at 340 nm and 380 nm.
Other methods
Cellular protein content was determined with a BCA protein assay
kit (Pierce, Rockford, IL). Data were statistically
analyzed by Student’s t test, using the Microsoft Corp. Excel computer program.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-4-isomerase and
cytochrome P45017
, lyase, respectively, because the
pregnenolone formation is the rate-determining step in overall
steroidogenesis (1, 2, 14). The acute stimulation of steroidogenesis
was estimated from the accumulation of pregnenolone during a 2-h
incubation after the application of NPS-ACTH or ACTH. The amounts of
accumulated pregnenolone increased with the dose of NPS-ACTH as well as
ACTH, and leveled off at a similar level (Fig. 1a
). The concentration required for
half-maximum stimulation (EC50) is about 1 nM
for NPS-ACTH, which is a hundred times higher than that for ACTH (10
pM).
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). On the other hand,
application of NPS-ACTH affected the cellular cAMP synthesis only
slightly. No stimulation of cAMP synthesis was observed on addition of
1 nM NPS-ACTH, upon which steroidogenesis was stimulated to
half maximal levels (Fig. 1b). Even on maximal stimulation of the
steroidogenesis by 100 nM NPS-ACTH, cAMP accumulation was
increased to only 5% of that in the presence of 1 nM ACTH.
Similar dose-responses on cAMP accumulation and steroidogenesis induced
by NPS-ACTH have been reported in rat and mouse adrenal cultured cells
(3). The results indicate that increase of cellular cAMP is not
essential for the stimulation of steroidogenesis by NPS-ACTH.
NPS-ACTH induced Ca2+ signaling
The effect of NPS-ACTH on the Ca2+ concentration in
individual cells was analyzed by video enhanced microscopic
fluorescence imaging. Application of NPS-ACTH induced typically three
types of Ca2+ signals in individual cells, i.e.
(a) series of Ca2+ spikes with a step-like Ca2+
elevation; (b) a step-like elevation of Ca2+ concentration;
(c) series of Ca2+ spikes without Ca2+
elevation (Fig. 2, a–c, respectively).
When the Ca2+ signals of 100–200 cells induced by 1
nM NPS-ACTH were analyzed in each experiment, signal types
in the cells were found to constitute 63 ± 5%, 28 ± 4%,
and 9 ± 2% of the relative population for (a), (b), and (c),
respectively (mean of four separate experiments). The frequency of
Ca2+ spikes was variable in individual cells (Fig. 2, a and
c), but it did not show apparent correlation with NPS-ACTH
concentration. The increase of the dosage of NPS-ACTH increased total
population of Ca2+ signaling cells without affecting the
signal height in individual cells. Similar tendency of Ca2+
signals was observed in the same cell system stimulated by 0.1–100
pM ACTH whose frequency and intensity were also independent
on the dosage of ACTH (12). The dose-dependence of percentage of total
number of Ca2+ signaling cells, (a) + (b) + (c), per number
of monitored cells on NPS-ACTH concentration corresponded well to the
curve for the stimulation of steroidogenesis up to 1 nM
NPS-ACTH (Fig. 3). In the presence of 10
nM of NPS-ACTH, 95 ± 4% of cells show
Ca2+ signals, but only 13 ± 1% in the absence of
NPS-ACTH (mean of four separate experiments). It must be noted that the
percentage of total Ca2+ signaling cells was almost
saturated around 1 nM NPS-ACTH, while steroidogenesis was
increased further with the increase of the peptide to 100
nM (Fig. 3). The action of NPS-ACTH above 1 nM
might be mediated by other second messenger(s) beside
Ca2+.
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indicating the voltage-dependent Ca2+ influx might be
essential for stimulative action of NPS-ACTH at these concentrations.
The concentration of nicardipine required for the complete suppression
of the stimulative effect of 1 nM NPS-ACTH was 40
µM in the culture medium containing 10% FCS. In the
serum-free medium, the stimulation of steroidogenic activity was
suppressed by 1 µM of nicardipine. The steroidogenic
activity of the cells in the serum-free medium, however, was
fluctuating in each experiment and a mean of the activities was about a
half of that in the serum containing medium. Therefore, we used serum
containing medium and 40 µM nicardipine to obtain clear
results in this experiment. Because nicardipine affected neither
nonstimulated nor 0.1 mM (Bu)2cAMP-stimulated
steroidogenesis (data not shown), the voltage dependent
Ca2+ influx might not be essential for the basal
steroidogenesis and cAMP-induced stimulation. The NPS-ACTH-induced
Ca2+ signals were also affected by the inhibitor. In the
cells pretreated with nicardipine, the Ca2+ elevation of
NPS-ACTH-induced (a) type Ca2+ signals disappeared
within a few minutes although the inhibitor has only a slight effect on
the quick rise in Ca2+ (Fig. 2d). Most of the
Ca2+ signaling cells (90 ± 5%, mean of three
separate experiments) showed Ca2+ increase only transiently
upon the addition of 1 nM NPS-ACTH to the nicardipine
pretreated cells. It can be concluded that the voltage-dependent
Ca2+ channels are responsible for the slow phase (>3 min)
of Ca2+ signaling which is essential for the stimulatory
action of NPS-ACTH in the steroidogenesis. When the cells were
stimulated by 10 and 100 nM NPS-ACTH, nicardipine did not
suppress the stimulative effect on steroidogenesis completely (Fig. 4a). The results indicate again that a second messenger(s) beside
Ca2+ affects the steroidogenesis in the presence of
NPS-ACTH above 10 nM.
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). The inhibitor completely suppressed
the stimulation in steroidogenesis induced by 1 and 10 pM
of ACTH, indicating that the Ca2+ influx is essential for
the action of ACTH at these concentrations. In the presence of
concentrations of ACTH higher than 100 pM, the inhibitory
action of nicardipine on the stimulated steroidogenesis was not
complete.
Suppression of the action of NPS-ACTH and ACTH by lipoxygenase
inhibitor
ACTH action on the steroidogenesis in bovine
fasciculata-reticularis cells is mediated by 15-lipoxygenase
metabolite(s) of arachidonic acid as a second messenger (14). The role
of the metabolite(s) in the action of NPS-ACTH could be elucidated by
treatment of the cells with AA-861, a lipoxygenase inhibitor. Addition
of AA-861 had no effect on the steroidogenesis stimulated by 0.1 and 1
nM of NPS-ACTH but suppressed to half the stimulative
effects by 10 and 100 nM NPS-ACTH (Fig. 5a). The role of 15-lipoxygenase pathway
on the stimulation of steroidogenesis is significant only at higher
NPS-ACTH concentrations, at which the percentage of total number of
Ca2+ signaling cells has already leveled off (Fig. 3). The
action of NPS-ACTH at 10 and 100 nM might be mediated by
lipoxygenase metabolite(s) together with Ca2+. By contrast,
the stimulative effect of ACTH on steroidogenesis was suppressed by
AA-861 at any ACTH concentration (Fig. 5b). Stimulation by 1
pM ACTH was completely suppressed by the inhibitor. These
results were consistent with a previous observation; 15-lipoxygenase
activity was stimulated by pM concentrations of ACTH
(14).
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and 5). The maximally stimulated steroidogenesis by
100 nM NPS-ACTH was completely suppressed by the coaddition
of the inhibitors (Table 1). Because
AA-861 did not affect 0.1 mM
(Bu)2cAMP-stimulated steroidogenesis (14) as like as
nicardipine, the data indicate that the second messengers for 100
nM NPS-ACTH were Ca2+ and lipoxygenase
metabolite(s) but not cAMP. In the case of ACTH-stimulated
steroidogenesis, the suppression induced by the coaddition of the
inhibitors was not complete upon the addition of 1 nM ACTH.
Because the ACTH at concentrations higher than 0.1 nM
stimulated cellular cAMP synthesis significantly (Fig. 1b), the action
of 1 nM of ACTH might be mediated by cAMP beside
Ca2+ and lipoxygenase metabolite(s).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Stimulation of
steroidogenesis by lower concentrations (<1 nM) of
NPS-ACTH might be mediated by increase of cellular Ca2+ as
a sole, or major, second messenger, because dose responses of the
percentage of total number of Ca2+ signaling cells and the
stimulation of steroidogenesis induced by NPS-ACTH were quite similar
(Fig. 3). Under the above conditions, nicardipine completely blocked
the stimulative action of NPS-ACTH on the steroidogenesis (Fig. 4a).
Upon the addition of higher doses (>10 nM) of NPS-ACTH,
the metabolite(s) of lipoxygenase activity might be important in
stimulation of the steroidogenesis together with Ca2+,
because the stimulation was partly suppressed by AA-861 (Fig. 5a).
Although a slight increase of cAMP synthesis was observed upon the
addition of 100 nM NPS-ACTH (Fig. 1b), the stimulation of
steroidogenesis was completely suppressed by pretreatment of
nicardipine and AA-861 at the same time (Table 1). Cellular cAMP can be
concluded not to be essential in the acute action of NPS-ACTH at any
concentration.
ACTH-induced acute stimulation of steroidogenesis is mediated by at
least three second messengers, cAMP, Ca2+, and lipoxygenase
metabolite(s), in bovine fasciculata-reticularis cells. Application in
the pM range of ACTH stimulated both Ca2+
signals and 15-lipoxygenase activity, while increase of total cellular
cAMP was too small to be detected by conventional methods (12, 13, 14).
Essential roles of Ca2+ and the lipoxygenase metabolite(s)
in the stimulative effect of ACTH on the steroidogenesis were confirmed
by the experiments using nicardipine and AA-861, both of which
inhibited the ACTH-induced stimulation without affecting basal
steroidogenesis. It must be noted that 0.1 mM
(Bu)2cAMP-stimulated steroidogenesis was not affected by
either of the inhibitors, indicating that the stimulative action of
ACTH at pM concentrations is not attributable to the
increase in cellular cAMP. Although no increase of cellular cAMP was
observed at 1 pM ACTH-stimulated condition, basal A-kinase
activity might be essential for steroidogenesis, which has been
indicated in A-kinase deficient adrenal tumor cells and in normal
adrenal cells where an A-kinase inhibitor, H-89, suppressed the basal
A-kinase activity (14, 20, 21). cAMP might be a second messenger in the
action of ACTH at concentrations above 10 pM, at which a
slight increase of cellular cAMP was observed (Fig. 1b). Upon the
addition of pharmacological concentrations of ACTH (>100
pM), cellular cAMP synthesis was significantly increased,
where the inhibitory effects of nicardipine and AA-861 on the
steroidogenesis were incomplete (Table 1). cAMP might be a significant
second messenger under these conditions, although Ca2+ and
lipoxygenase metabolite(s) still have roles in the action. It has been
reported for the chronic effect of ACTH in the same cell system that
cAMP does not function as a sole intracellular mediator of induction of
steroidogenic enzymes, even upon the application of very high
concentrations of ACTH, 1 µM (22).
In the bovine fasciculata cells, voltage-dependent Ca2+ channels have been predicted to function in stimulation of steroidogenesis (13). Enyeart et al. (9) reported that T-type rather than L-type Ca2+ channels are required for ACTH-induced stimulation of steroidogenesis. They found with patch clamp experiments that T-type Ca2+ channels operate in ACTH action with an EC50 of 10.4 pM (8). The concentration of the EC50 is consistent with our previous observation that showed the percentage of Ca2+ signaling cells was increased by 0.1–100 pM of ACTH (12). Ca2+ is one of the major second messengers in angiotensin II or potassium-induced stimulation of aldosterone synthesis in adrenal glomerulosa cells (23). The T- and L-type Ca2+ channels in the bovine glomerulosa cells were also completely blocked by nicardipine, which abolished potassium-induced stimulation of aldosterone synthesis (24). The frequency of series of Ca2+ spikes in the glomerulosa cells, however, was significantly increased by the increase in concentration of angiotensin II, although that in fasciculata-reticularis cells was not correlated with the concentration of NPS-ACTH or ACTH (25). The Ca2+ signaling mechanism in fasciculata-reticularis cells, which was stimulated by ACTH or NPS-ACTH might not be the same as in the glomerulosa cells induced by angiotensin II and potassium.
The Ca2+ and 15-lipoxygenase metabolite(s) are second
messenger for ACTH and NPS-ACTH induced stimulation in the bovine
adrenal cells. Interaction of those two second messenger systems has
been reported in various cells. Cellular Ca2+ signals were
reported to activate phospholipase A2, which stimulated
release of arachidonic acid in adrenal cells, and also to activate
cytosolic 15-lipoxygenase activity in human polymorphonuclear leukocyte
(26, 27). Addition of lipoxygenase metabolites of arachidonic acid has
been reported to stimulate Ca2+ releases from mitochondria
that were isolated from the bovine zonae fasciculata-reticularis and
also from rat liver cells (28, 29). In the bovine
fasciculata-reticularis cells, there is a possibility of cross-talk
between Ca2+ signals and the lipoxygenase pathway in the
ACTH-induced stimulation because the stimulation was inhibited by
nicardipine and AA-861 at the same ACTH concentrations, indicating the
Ca2+ and 15-lipoxygenase metabolite(s) function at the same
time (Figs. 4b and 5b). On the other hand, NPS-ACTH-stimulated
steroidogenesis was inhibited by nicardipine and AA-861 at different
NPS-ACTH concentrations (Figs. 4a and 5a). These data might indicate
Ca2+ and 15-lipoxygenase metabolite(s) stimulate
steroidogenesis independently. The interaction between Ca2+
signals and the lipoxygenase pathway in the bovine
fasciculata-reticularis steroidogenesis might not be simple and remain
to be clarify.
Only one class of ACTH-receptor has been identified in adrenal fasciculata-reticularis cells (30, 31). NPS-ACTH might bind to the ACTH receptor because ACTH-induced cAMP synthesis was antagonized by NPS-ACTH (32, 33). Truncated ACTH, ACTH11–24, antagonized ACTH-induced cAMP synthesis in mouse ACTH receptor expressed in HeLa cells (34). The IC50 of the truncated ACTH was about 1 nM, similar to EC50 of NPS-ACTH for the stimulation of the steroidogenesis in this study. The ACTH11–24 could stimulate steroidogenic activity in rat adrenal cells (35). Because the NPS-ACTH is modified at Trp9 of ACTH1–24, the nitrophenyl sulfenyl residue might prevent binding of the amino-terminal region of ACTH to the specific site in the ACTH receptor. The remaining region of ACTH11–24 might stimulate steroidogenesis without increase of cAMP.
For investigation of the mechanism for Ca2+-mediated stimulation of steroidogenesis in the fasciculata-reticularis cells, ACTH-induced stimulation might not be suitable because the stimulative action of ACTH is mediated by not only Ca2+ but also other second messengers at any ACTH concentration. On the other hand, Ca2+ is a solo, or major, second messenger for the action of 1 nM NPS-ACTH at which steroidogenesis was stimulated to a half maximum level. The NPS-ACTH-induced Ca2+ signals were indistinguishable from those induced by ACTH in terms of both signal shape and intensity. Therefore, NPS-ACTH-stimulated cultured cells might be a suitable experimental system for investigating the mechanism of stimulation of steroidogenesis by Ca2+ signals. Further experiments on Ca2+-induced cellular responses are underway in our laboratory.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Biotechnology and Life Science,
Faculty of Technology, Tokyo University of Agriculture and Technology,
Koganei, Tokyo 184, Japan.
Received April 8, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, lyase-mediating pathway of androgen
synthesis in bovine adrenocortical cultured cells. Biochim Biophys Acta 1134:143–148[Medline]
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