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Baker Medical Research Institute, Melbourne, Australia
Address all correspondence and requests for reprints to: Prof. John W. Funder, Baker Medical Research Institute, P.O. Box 348, Prahran, Victoria Australia 3181.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1
nM aldosterone, with steroids showing a hierarchy of
potency aldosterone = 9
fluorocortisol >
deoxycorticosterone > corticosterone > spironolactone. Both
Ca2+-dependent and -independent PKC activity appear equally
inhibited by aldosterone, and PMA-stimulated increases in PKC activity
appear similarly aldosterone-sensitive. No displaceable binding of
[3H]aldosterone to purified PKC can be shown, evidence
against a direct effect of aldosterone on PKC; aldosterone does not
alter basal or PMA-stimulated PKC activity in cardiac fibroblasts,
evidence for a cell-specific mediator of the myocyte effect. Taken with
the previous demonstration of the potentiation of aldosterone-specific
MR-mediated effects by PKC activation, the present data argue for the
existence of a complex cross-talk mechanism between aldosterone and
factors affecting PKC activity in the heart. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently shown that MR occupancy by aldosterone (but not corticosterone) increases [3H]leucine incorporation into protein by isolated neonatal rat cardiomyocytes, and that this effect is potentiated by elevated extracellular glucose (8). In contrast with MR occupancy by aldosterone, glucocorticoid receptor (GR) activation is catabolic, lowering [3H]leucine incorporation, an effect that is not altered by high glucose. The glucose-induced potentiation of aldosterone action is totally blocked by the selective protein kinase C (PKC) inhibitor GF 109203X, which strongly suggests a link between PKC activity and the action of aldosterone, but not glucocorticoids, in cardiomyocytes.
In terms of a more global relationship between these signaling pathways, other recent studies have also shown the involvement of PKC as a possible second messenger in the nonclassical actions of aldosterone. Aldosterone has been reported to stimulate PKC activity in rat distal colon (9), and to translocate PKC from the soluble to the particulate fraction in rat vascular smooth muscle cells (10), in each case with a time course of action inconsistent with an effect via classic genomic pathways. Modulation of PKC by aldosterone may thus represent an additional important mechanisms underlying selective aldosterone action.
Activation of PKC is generally associated with translocation of the
enzyme from the soluble to the particulate fraction, which is thus
often used as an index of activation; in this context, translocation of
PKC in response to various hormones and peptides has been documented in
neonatal rat cardiomyocytes (11, 12). There is also, however, evidence
that translocation of PKC does not always correlate with enzyme
activation as measured by substrate phosphorylation (13); for example,
PKC isoform 
does not translocate even when stimulated by phorbol
esters (14). This suggests that measurement of net PKC substrate
phosphorylation may constitute a more accurate index of PKC activation
than translocation, and we have therefore chosen to determine
phosphorylation in vitro to evaluate PKC activity in the
present study. We have thus examined the effect of aldosterone and a
various other steroids on PKC activity in neonatal rat cardiomyocytes,
to further explore the actions of aldosterone in this nonepithelial
tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fluorocortisol, spironolactone, phorbol 12-myristate 13-acetate
(PMA), and DMEM were from Sigma (St. Louis, MO).
[
-32P]adenosine 5'-triphosphate (ATP) was from
Bresatec (Adelaide, Australia). [1,2,6,7-3H]aldosterone
was from DuPont-New England Nuclear Research Products (Boston, MA).
Ham’s F-12 medium and nonessential amino acids were from ICN
Biomedicals (Sydney, Australia). RU 28318, a type I (mineralocorticoid)
receptor antagonist, was provided by Roussel-UCLAF (Paris, France). All
other chemicals were of the highest purity commercially available.
Steroid hormones were dissolved in ethanol, and the final concentration
of ethanol in all assays was less than 0.01%, at which concentration
ethanol was without effect on PKC activity in our assay system.
Cell culture
Enriched cultures of neonatal (day 1–3) cardiomyocytes were
prepared from hearts of Sprague-Dawley rats by a previously described
enzymatic method (8). After treatment with collagenase type II (Sigma),
and subsequent digestion with trypsin and deoxyribonuclease, cells were
resuspended in complete medium [50% (vol/vol) DMEM, 50% (vol/vol)
Ham’s F-12 medium, 1% nonessential amino acids, 2 mM
L-glutamine, penicillin (100 U/ml), streptomycin (100
µg/ml)] containing 10% FCS, and incubated for 40 min at 37 C,
allowing selective attachment of nonmyocytes. Cardiomyocyte-enriched
suspensions were plated in complete medium containing 10% FCS. The
culture medium was changed to complete medium with 10% FCS containing
100 µM of the DNA synthesis inhibitor bromodeoxyuridine
(BdU) on the second day in culture, and kept at 37 C in a humidified
atmosphere of 5% CO2 in air for a further 2 days. Each
dish was incubated with complete serum-free medium [50% (vol/vol)
DMEM, 50% (vol/vol) Ham’s F-12 medium, 1% BSA (BSA)] for 16 h
before experiment. Using this method, we routinely obtained
myocyte-rich cultures with at least 95% cardiomyocytes, as assessed by
microscopic observation of beating cells and immunofluorescence
staining with a monoclonal antibody against atrial natriuretic
factor.
Most nonmyocyte-enriched cultures consist of cardiac fibroblasts, which were then cultured in DMEM containing 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and the medium changed every 3 or 4 days. Cardiac fibroblasts between the second and fourth passages were used, after having been incubated with serum-free DMEM for 16 h.
Determination of PKC activity
Preparation of cardiomyocyte soluble and particulate fractions
was by a previously described method (8). Cells were washed twice with
ice-cold PBS, and scraped into ice-cold buffer I containing 0.25
M sucrose, 10 mM MOPS, 2.5 mM EGTA,
2.0 mM EDTA, 100 µM leupeptin, and 200
µM phenylmethylsulfonyl fluoride. The cells were
sonicated twice for 10 sec on ice, and centrifuged at 100,000 x
g for 60 min at 4 C, with the supernatant taken as soluble
fraction. The pellet was resuspended in buffer I, solubilized with
0.5% (vol/vol) Tween-20 for 30 min on ice, sonicated, centrifuged at
100,000 x g for 60 min at 4 C, and the supernatant
taken as the particulate fraction. Protein levels were determined by
the method of Bradford (Pierce Chemical, Rockford, IL) with BSA as
standard. PKC assays were performed at 30 C in a final volume of 40
µl of incubation mixture containing either soluble or particulate
fraction with 30 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 200 µM CaCl2, 200
µM [-32P]ATP, 100 µg/ml or 50 µg/ml
myristoylated alanine-rich C kinase substrate (MARCKS), 10 µg/ml
phosphatidylserine, and 10 µg/ml diolein, as previously described
(15). Parallel incubations performed in the absence of
phosphatidylserine and diolein were also included. Reactions were
allowed to proceed for 15 min, except in time-course studies, and
incubations stopped by adding 8 µl of 375 mM
orthophosphoric acid at 4 C. Subsequently, 30 µl aliquots of the
reaction mixture were spotted onto 2 x 2 cm squares drawn on a
Whatman P-81 phosphocellulose paper and then washed once for 10 min in
75 mM orthophosphoric acid, twice for 5 min in distilled
water and then for 5 min in 95% ethanol. After washing, the paper was
dried, cut into individual squares, placed in individual scintillation
vials, and radioactivity counted in a scintillation spectrometer. PKC
activity was expressed as picomoles of Pi/min/mg of protein.
Purification of PKC
PKC was purified to homogeneity from adult Sprague-Dawley rat
brains by a previously described method (16), made 10% with glycerol
and 0.05% with Triton X-100, and stored at -80 C. This PKC
preparation has been shown to be very active in phosphorylating MARCKS
in the presence of Ca2+ and phosphatidylserine
(Ca2+/phosphatidylserine-dependent), and to be activated by
PMA in a concentration-dependent manner (16).
Binding assay
[3H]Aldosterone binding to purified PKC was
assessed in two independent binding assays. Purified PKC was incubated
with 0.5–10 nM [3H]aldosterone (84.8
Ci/mM) alone or with excess (1 µM)
aldosterone in binding buffer (50 mM Tris-HCl, pH 7.4, 1
mM MgCl2, 1 mM CaCl2, 5
mM KCl) for 90 min at 27 C, and then filtered onto a 0.2
µm nitrocellulose membrane (Schleicher & Schuell, Keene, NH) in a
slot-blot transfer apparatus. After washing with 50 mM
Tris-HCl three times under vacuum, the membrane was removed and air
dried. The dried membrane was exposed to a BAS-TR2040S imaging screen,
and then analyzed by phosphoimage analysis (BAS, Fuji, Tokyo, Japan),
and/or cut into individual pieces and the radioactivity counted.
Overlay-binding assay was performed by a previously described method
(17); different concentrations of purified PKC were blotted onto a
nitrocellulose membrane in a slot-blot transfer apparatus, washed twice
for 5 min in 5 mM imidazole (pH 7.4), and then incubated in
a solution containing 5 mM imidazole, 60 mM
KCl, 5 mM MgSO4, and 1 µCi/ml
[3H]aldosterone, pH 7.4, for 30 min at room temperature.
The membrane was washed twice in 30% ethanol/H2O for 10
min, and then [3H]-labeled bands were analyzed by BAS
phosphoimage analysis.
Statistical analysis
Data are expressed as mean ± SEM, and
statistical significance assessed by one-way ANOVA followed by
Fisher’s PLSD test for post hoc comparisons, with
P values of < 0.05 taken as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Given
the much higher basal levels of PKC activity in the soluble fraction,
and the greater relative repression by aldosterone (Fig. 1), we have
chosen this fraction for further studies. Figure 1B shows the time
course of the effect of aldosterone (solid circles) and PMA
(open squares) on PKC activity. Soluble fractions were
incubated for the time indicated with 1 nM aldosterone or
100 nM PMA, and PKC activity measured. PMA produced a
significant increase in PKC activation after 2 min incubation (125
± 12% of control; P < 0.05), and reached plateau
levels at 15–20 min. In contrast, aldosterone very rapidly lowered
basal PKC activity in the same cultured cells within 1 min (75 ±
2% of control; P < 0.05), reaching 52 ± 12% of
control at 15 min.
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shows the effect of different concentrations
of aldosterone on basal PKC activity. Soluble fractions were incubated
with the indicated concentration of aldosterone for 15 min and PKC
activity measured. Lowering of PKC activity became apparent at 1
nM aldosterone, reaching a plateau at 10 nM. A
similar dose-response study was performed to explore the effects of
corticosterone on PKC activity in neonatal cardiomyocytes. As shown in
Fig. 2B, corticosterone was without effect on PKC activity at
physiological plasma free concentrations, but lowered basal PKC
activity at higher concentrations (100 nM-1
µM). These results suggest that aldosterone can
selectively repress basal PKC activity in neonatal rat cardiomyocytes,
in that corticosterone is almost two order of magnitude less potent in
this regard.
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, the potent mineralocorticoid 9-fluorocortisol
represses basal PKC activity to a similar extent as aldosterone. The
same concentration (10 nM) of deoxycorticosterone has
considerably lower potency in terms of repressive effects on basal PKC
activity; estradiol has a minimal but significant effect, and
progesterone was without effect. The dose-response curve for
9-fluorocortisol was similar to that of aldosterone, with a
substantial effect on basal PKC activity at 1 nM; in
contrast, a significant effect for deoxycorticosterone was first seen
at 10 nM (data not shown).
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, 100 nM PMA produced a significant increase in
PKC activation after 15 min incubation (P < 0.01
vs. basal value). Coincubation with aldosterone at as low as
1 nM lowered PMA-stimulated PKC activity, an effect of
aldosterone that is dose dependent (Fig. 4). The
dose-response curve for aldosterone on PMA-stimulated PKC activity is
clearly parallel to that for basal PKC activity, with plateau levels of
repression at aldosterone concentrations of 
(Fig. 2); however,
even at high concentrations of aldosterone, PKC activity is not
repressed to the level seen with 10 nM aldosterone alone
(compare PMA + 10 nM Aldo with Aldo 10 nM
alone). On the other hand, the percent fall in PMA-stimulated PKC
levels with aldosterone (26%) is indistinguishable from that seen with
10 nM aldosterone in the absence of PMA (29%), suggesting
equivalent sensitivity of basal and PMA-stimulated PKC activity.
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, addition
of Ca2+ (200 µM) more than doubles the level
of PKC activation compared with phosphatidylserine and diolein alone.
These results are consistent with earlier studies (11, 18) showing that
in neonatal rat cardiomyocytes Ca2+-sensitive PKC isozymes
are expressed. In the present studies, Ca2+-dependent-PKC
activation was observed over the 25–200 µM
Ca2+ concentration range (Fig. 5B, open
circles). Aldosterone (10 nM; Fig. 5B, closed
circles) repressed Ca2+-dependent-PKC activation, with
a similar degree of repression at each Ca2+-concentration
(61 ± 2% at 200 µM Ca2+, 66 ±
2% at 100 µM, 70 ± 1% at 50 µM,
66 ± 2% at 25 µM). These data suggest that
inhibitory effect of aldosterone on PKC activity is essentially
independent of Ca2+ concentration, and that in contrast
with the case for PMA, both Ca2+ independent and
Ca2+ dependent isozymes are similarly aldosterone
sensitive.
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, no
specific binding was seen. Secondly, various concentrations of purified
PKC (880 ng-44 µg) were spotted directly onto a nitrocellulose
membrane, and incubated with a solution containing 1 µCi/ml
[3H]aldosterone, and
[3H]aldosterone-binding was analyzed by phosphoimaging.
Although we have previously shown that this purified PKC can directly
bind 45Ca2+(17), no specific binding (which
would show as a dark line in the slot) was seen with
[3H]aldosterone (Fig. 6B). These results suggest that the
involvement of a protein intermediate in the inhibitory action of
aldosterone on PKC activity.
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, spironolactone at low concentrations was without
effect on basal PKC activity; at 100 nM, 1
µM, spironolactone repressed basal PKC activity but to an
apparent plateau level significantly higher than that seen with
aldosterone. Of particular interest, 1 µM spironolactone
did not ameliorate the effect of 10 nM aldosterone, which
might be expected if the two steroids were competing for a common
binding site on MR. We have also used the more selective MR antagonist,
RU 28318, and found similar results to those shown for
spironolactone.
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, basal PKC activity was
much less in cardiac fibroblasts (116 ± 10 pmol/min·mg) than in
cardiomyocytes. Aldosterone (1–100 nM) was without effect
on basal PKC activity in cardiac fibroblasts, as were the same
concentrations of deoxycorticosterone and 9-fluorocortisol. PMA (100
nM) produced a near doubling of PKC activation (224 ±
8 pmol/min/mg, P < 0.01 vs. basal), and
aldosterone (10 nM) had no effect on PMA-stimulated PKC
activity. These results argue that inhibitory effect of aldosterone on
PKC activity is cardiomyocyte specific, given that aldosterone clearly
does not affect PKC activity in cardiac fibroblasts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PKC comprises a family of serine-threonine protein kinases, which have
a number of key cellular functions (20, 21). In the heart, PKC has been
implicated as a candidate mediator of cardiomyocyte hypertrophy, and of
contractility and ion exchange activity (22). Whereas PKC activity is
regulated physiologically by membrane receptor-mediated production of
diacylglycerol, and experimentally by phorbol esters such as PMA, there
are an increasing number of reports of steroids as effectors in the PKC
system. At the level of gene expression, thyroid hormone has been shown
to repress PKC expression in neonatal heart, and PKC
in both
neonatal and adult heart (23). At the other end of the scale, purified
enzyme preparations of PKC, PKC and PKC have been reported to
be directly activated by 1,25 dihydroxy vitamin D3, in a
system containing only purified enzyme, substrate, cofactors and lipid
vesicles (24). The concentrations of vitamin D3 required in
the above study were 10–100 nM, approximately three orders
of magnitude higher than those required for binding to classical
vitamin D3 receptors and producing genomic effects.
Effects of aldosterone on the PKC system have also been reported. In
previous studies from this laboratory (3), the rapid nongenomic effects
of aldosterone on intracellular Ca2+ in vascular smooth
muscle cells were modulated by PMA and staurosporine, suggesting
participation of PKC in the signaling process. In the rat distal colon
(9), aldosterone has been reported to increase PKC activity greater
than or equal to 10-fold over basal in cytosolic fractions within 15
min; 9-fluorocortisol produced a similar level of increase, whereas
deoxycorticosterone (5-fold), estradiol (2- to 3-fold), and cortisol
(1.5- to 2-fold) showed lower levels of activation over basal.
Interpretation of these data are difficult, in that for all steroids
tested indistinguishable levels of stimulation were found over the
concentration range 10 pM to 100 nM. The
potential physiologic role for such an effect independent of dose over
the range of normally circulating concentrations remains to be
established.
Whereas the steroid concentration required for the 1,25 dihydroxy
vitamin D3 effect (24) appear orders of magnitude higher
than circulating levels, and those for modulation of PKC activity in
colonic cells (8) orders of magnitude lower, the dose-response data
found in the present study are compatible with a graded cellular
response to changes in plasma concentration over the normal physiologic
range. For aldosterone, the EC50 for effects on
cardiomyocyte PKC activity lies between 0.1 and 1 nM, in
line with previous studies by Wehling and his colleagues on rapid,
nongenomic effects of aldosterone in a variety of tissues (25). The
hierarchy of steroid potency is similar but not identical to that found
in such studies, with 9-fluorocortisol essentially equivalent to
aldosterone, and deoxycorticosterone active but requiring rather higher
concentrations. Where the present study differs in this regard is that
previously (25) the physiologic glucocorticoids were found to be devoid
of agonist activity, even at concentrations 1000x those of aldosterone,
and spironolactone similarly without aldosterone antagonist activity.
In the present study, in contrast, corticosterone mimics aldosterone,
although requiring two orders of magnitude higher concentrations to do
so, and, at similarly high concentrations, spironolactone appears to
act as a partial agonist for the aldosterone induced response, with no
apparent antagonist activity, a finding difficult to reconcile with
current models of partial agonist/antagonist action.
The question to be addressed, in the context of these steroid
specificity studies, is that of how the nanomolar concentrations of
aldosterone are recognized, and the mechanism whereby that recognition
is transduced into a signal affecting PKC activity. The nanomolar
concentrations at which the effects of aldosterone are seen require a
high affinity recognition system, which presumably involves binding to
protein. We have shown that purified PKC preparations do not bind
aldosterone with high affinity, a finding extrapolated to the whole
cell milieu with some caution; at first sight, the present specificity
data might be interpreted as excluding a rapid, nongenomic action for
classical MR, leaving open the possibility of aldosterone acting by
binding to some other membrane or intracellular
protein/channel/effector. On the other hand, it may be premature to
exclude protein-protein interactions involving classical MR as
mediating the rapid effects of aldosterone and the other steroids
examined on cardiomyocyte PKC activity. Such protein-protein
interactions have been demonstrated for GR with both AP-1 and NF
B
protein complexes (26, 27, 28). Secondly, whereas classical MR have been
widely reported to have very similar affinity for aldosterone and the
physiologic glucocorticoids (29, 30), in transfection system
aldosterone has been reported to be equivalent to (31), ten times more
potent than (32) or even 100 times more potent than physiologic
glucocorticoids (33), with the mechanism underlying the differences in
observed potency yet to be established. Finally, in epithelial MR
corticosterone and cortisol mimic the action of aldosterone, whereas in
nonepithelial MR the physiologic glucocorticoids act, like
spironolactone, as aldosterone antagonists (34); again, the basis for
this difference is unclear. In the final analysis, a possible role for
classical MR in the rapid PKC response will be excluded or established
by studies in the mineralocorticoid receptor knockout mouse,
anticipated to be available in the relatively near future.
In previous studies we have shown that high glucose elevates PKC activity in neonatal cardiomyocytes, and that this elevation of PKC has profound effects on MR-mediated actions of aldosterone in these cells (8). Specifically, the threshold for aldosterone action (on [3H]leucine incorporation into protein) is lowered by an order of magnitude, thus very much enhancing the sensitivity of the cell to ambient concentrations of steroid. The effect on MR is aldosterone-specific, in that it is not seen when MR are occupied by corticosterone, and abrogated by the selective PKC inhibitor GF109203X. The present studies suggest that an intracellular negative feedback control system may operate for PKC and aldosterone in these nonepithelial cells, with aldosterone rapidly but modestly lowering PKC activity, and PKC activation increasing by an order of magnitude the potency of aldosterone at the genomic level, on at least one index of gene expression. The mechanisms involved in this cross-talk between the two signaling systems, and the pathophysiologic implications thereof, await further investigation.
Received February 14, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activated by phorbor esters, epinephrine, and endothelin. Circ Res 72:757–767, 25-dihydroxyvitamin D3. J Biol Chem 270:6639–6643B in
mediation of immunosuppression by glucocorticoids. Science 270:283–286B
activity through induction of IkB synthesis. Science 270:286–290This article has been cited by other articles:
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