Vol.
89,
July
12, 1979
No.
1, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
COMMUNICATIONS
Pages
PARADOXICAL NATURE OF MINERALOCORTICOID +M. K. Agarwal
May 25,
77-84
RECEPTOR ANTAGONISM BY PROGESTINS
and
++
+CNRS and ++ UER Broussais-Hztel Received
RESEARCH
J. Paillard Dieu,
Paris,
France
1979
SUMMARY: Progesterone, but not the synthetic progestagen R-5020, was as good as aldosterone in displacing the mineralocorticoid from its specific receptor in rat kidney both in classical competition studies and in aldosterone binding to MRl and MR2 components of MR during physical separation. Paradoxically, at equimolar (10-S M) concentrations, both gestagens (progesterone = 1, R-5020 = 20) were preferentially bound to the MR4 component which coeluted with serum bound 14C-corticosterone (or 14C-progesterone), which could not be labelled with aldosterone in the kidney, and which could not be detgcted in the liver and the serum under any condition. The MRl entity, at loM, wa:s saturated as: aldosterone = 2, R-5020 = 1 = progesterone. Thus, the agonist and the antagonist function in mineralocorticoid action may be expressed by two or more distinct and different sites. INTRODUCTION: their
target
to nuclear this
specific
bits agoist
only
or via
mediated
binding
in competition
vector,
receptor
we have
corticoids for
an "ideal" recently
Abbreviations glucocorticoid
five
synthetic
of the
either
cellular
vector
(2).
diuresis
(3),
and antagonises
(4).
Contrary
polymorphism separation
of steroid (6),
R-5020
tritiated
form,
by an antihormone that
does not
MR = mineralocorticoid receptor.
disease,
of receptor
for
and this (1).
may proceed bind
of a
mineralo-
seems true
In the
(promegestone was used
inhireceptor-
hypothesis
hormones
complex
Proges-
to the
(5),
to
by reversing
Addison's
progestin in the
may act
to bind
with
physical
in the kidney
used are: specific
translocation
another
studies
classes
appear
in patients
demonstrated
antagonism
subpopulation
with
natriuretic
synthesized
mineralocorticoid that
all
subsequent
effects
during
hormones
'Antihormones'
association
aldosterone
corticosteroid
with
(1).
aldosterone
and gluco-
study,
sites
opposes
unitary
the
step,
receptor
acceptor
sequence
terone
In an initial
present 17-a--methyl),
to demonstrate via
of
that
saturation
of
aldosterone.
specific
receptor;
+Address after 15 July 1979: 15 Rue de 1'Ecole de MGdecine, 75270 Cedex 06, France, to whom all correspondance should be sent.
GR = Paris
0006-291X/79/130077-08$01.00/0
77
Copright All rights
@ 1979 by Academic Prrss. Inc. in unyform resrrved.
of reproduction
Vol.
89,
No.
BIOCHEMICAL
1, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
MATERIALS AND METHODS: Male, Wistar rats (150-200 g) were bilaterally adread libitum nalectomised 2-3 days prior to use; food and 1% NaCl were provided after surgery. Animals were sacrificed under ether anaesthesia, the desired organ was perfused with the initial buffer, according to the experimental procedure, and cell sap was finally obtained by centrifugation at 105,000 g (50 min). Blood, obtained by aortic cannulation was allowed to clot at 37°C (30 min), then at 4°C (60-90 min), and finally centrifuged at 3000 g. For competition and association studies, 0.5 ml cytosol (in 0.01 M Tris(60 min 4'C) in presence of a desired HCl, pH 7.4), or serum, was incubated amount of the radioactive steroid alone or in presence of a known excess of the cold homologous or heterologous molecule. Free steroid was removed by the addition of 0.5 ml (50 mg/ml) of activated charcoal (Sigma C-5260), further incubation (10 min 4"C), and centrifugation (15 min, 4000 g). Aliquots of 0.5 ml were mixed with 10 ml Scintix (Isotec, France) and counted in a Packard Tricarb Scintillation Spectrometer (1,2,5). Protein was quantitated by the Biuret method and all optical densities were read manually. organ cytosol, prepared in the initial buffer as per For chromatography, was equilibrated with the steroid and charcoal treated as elution procedure, above,and loaded onto the resin as described previously (1,2,5). Undiluted serum was similarly treated; further details are given in appropriate legends. 1,2,3H-aldosterone (53 Ci/m mol; lot 32), 1,2,6,7,3H-progesterone (95 Ci/ m mol; lot 26), 4- 14C-corticosterone (52 mCi/ m mol; lot 12), and 4-14Cprogesterone (61 mCi/m mol; lot 38) were purchased from Amersham (U.K.). 3HR-5020 (promegestone 17-a-methyl; 87 Ci/m mol; lot 1009-149) was a product of New England Nuclear (USA). Radiochemical purity exceeded 98% in all cases. RESULTS AND DISCUSSION:
Data
aldosterone
in both
targets
was similar of mineralocorticoid . to exhibit
appeared heart the
steroid; Data
and the to vectors
diluted
(l/10) in Fig.lc
displacing effective
establish
In a reciprocal kidney
bound
three
steroids
data
was far no binding
confirm
progesterone
regard, experiment
3H-R-5020. (aldosterone,
that from
its
(not In view
for
shown),
cold
progesterone,
78
(Fig.
whereas other
of receptor
in the
could
be of
R-5020
liver,
whereas
lb).
R-5020
synthetic
aldosterone multiplicity R-5020)
was far gestagens
very
poorly
(1,2,5) bind
of
(3,4).
was as good as aldosterone
receptor,
as reported
to the dose
Furthermore,
than
at all
the
characterstics
la vs lb).
greater
serum exhibited
in
of R-5020
association
(Fig.
binding
literature
quantities
of
two known
proportional
in the
greater
the
characterstics
bound
comparable
in the kidney
3H-aldosterone in this
was
progestin
of binding
progesterone
association
far
despite
"ideal"
However,
earlier
that
the kinetics
and the kidney,
(1).
different
confirm
cytosol,
binding
Data
mostly
in Fig.lb
aldosterone
heart
Serum aldosterone
these
to kidney
the
show that
hormones
somewhat
vs the kidney.
bound
in Fig.la
the
in less (3,4). displaced
, do these
same vector?
Vol. 89, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
t
STEROID
CONCENTRATION
(M)
Fig.
1. ASSOCIATION CHARACTERSTICS OF STEROID BINDING IN SEVERAL RAT TISSUES For Fig.la, renal cytosol was equilibrated with 3H-aldosterone (X) or 3Hprogesterone (A), myocardial with either 3H-aldosterone (0) or 3H-progesterone (a), and serum with 3H-aldosterone (X-----X). For Fig.lb, renal (X) or hepatic (C)) cytosol was incubated with 3H-R-5020. For Fig.lc, renal cytosol in presence of 10-8 M 3H-aldosterone was put in contact with the indicated concentration of non-radioactive aldosterone (O), progesterone (a), or R-5020 (X ). Non-specific binding in all cases was calculated by determination of radioactivity in presence of one thousandfold excess of cold, homologous steroid and was subtracted from the corresponding value obtained with tritiated hormone alone. Each sample (cold + radioactive or radioactive alone) was studied in triplicate at each point shown in the figure. For other details seeMethods and earlier publications (1,2,5).
Results MRl, Even
in Fig.2a
MR2 and GR type 10
-7
establish
of receptors
Pi progesterone
labelling
did
coeluted results
with
serum
transcortin
were
obtained
with
progesterone
in scale).
of
ralocorticoid
from
(3,4);
however,
logical
range,
than
its
action specific
at equimolar aldosterone
progestagen
progesterone
cytosol
to all
unlabelled
(Fig.2c)
progesterone
receptor
saturates
(please
components
can proceed
( 1o-8
M) binding
transcortin
via
in the
elution.
2a vs 2b).
bound
79
More
Similar
where
qunatitatively
note
the difference
in Fig.2a
diminished
(confirming
Fig.lc),
of the mine-
of progestagen
or 10 -7 M) concentrations,in a specific
the
to MR4 which
(Fig.Zb).
displacement region
to
MR2 whereas
(Fig. were
in the kidney
binding
higher
of aldosterone
aldosterone
myocardium
aldosterone 100 fold
with the
of
-8
aldosterone-specific
(T) bound
was bound
Since
in presence antagonism
of
(10
T = position
label
than
huge quantities
aldosterone
(5);
not
of MRl was greater
importantly,
less
further
MR2 and progeserone
the
excess physiothe MR4
Vol.
89,
No.
1, 1979
BIOCHEMICAL
GR
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
T
1
FRACTION
Fig.
FRACTION
NUMBER
NUMBER
FRACTION
NUMBER
2. ION EXCHANGE SEPARATION OF STEROID BINDERS ON DEAE-5'2 COLUMNS
5 ml renal cytosol was incubated with 10e8M 'H-aldosterone (a); 4 ml of either kidney (b) or myocardium (c) cytosol was incubated with 10e7M 3H-proin presence of 0.5 uCi of y;:E;z; ,E;zn;ig. *b 9 2 ml serum was incubated ; all samples were charcoal treated (100 mg/ml mixture), and loaded onto DE-52 columns (1 x 25 cm). separately, mixed for Fig.Zb, After a low ionic prewash (0.001 M phosphate, pH 7.5), elution was begun (at the arrow) with a gradient between 60 ml each of 0.001 M and 0.2 M of this buffer, pH 7.5. 1 ml samples were processed for radioactivity and the absorbance was recorded manually; further details in Methods and (1,2,5).
--------------A
which
is not
(see
below) Five
280;
0-0
transcortin and,
fold
first
second,
greater -7
3W
quantities
(10
reversed
MRl was considered
kidney
if bound
because
because
when R-5020
M) was used
R-5020
of
in place (Fig.
obtained
when undiluted
cytosol
and
was neither
excess
cold
coelutes too,
eluted
nonspecific
with
R-5020)
nor
MR4 (Fig.3a in the (displaced
coincided vs 3b).
same position by excess
14C
does not
competes
label
with
radioactivity
in the MR4 region
this
R-5020
R-5020
of radioactivity (Fig.3b)
0-0
are
Furthermore,
80
the
serum-R-5020 R-5020),
order
of
a small
peak
in place
of renal
in presence
14 C-corticosterone bound
was
quantities
with
(abolished
liver
MR4.
in the MR4 region
The tremendous
serum bound
of cold
for
to be contrasted
specific
as the
progesterone
but
serum was used
with
carrier
eluted
of progesterone, 3a vs 2b).
this
R-5020
of which
(Fig.3c),
complex
(Fig.3b),
was 50 times
less
was than
MR4
Vol.
89,
No.
1, 1979
BIOCHEMICAL
0-3)3Hfkh0-‘)
AND
EIOPHYSICAL
RESEARCH
COMMUNICATIONS
a
b
2
P ‘z -0 FRACTION
Fig.
NUMBER
FRACTION
NUMBER
3. ION EXCHANGE SEPARATION OF R-5020
FRACTION
NUMBER
BINDERS ON DEAE-CELLULOSE-52
4 ml renal cytosol (a), undiluted serum (b), or liver cytosol (c) was incubated with 10e7 M 3H-R-5020; 2 ml fresh serum with 0.5 PCi 14C-corticosterone was used for cochromatography as for Fig. 2 and (1,2,5). --------------A
-
280;
bound
R-5020
in the kidney
transcortin R-5020
was less
terone serum
than
(Fig.3b)
= 20,
and the
via
revealed
not liver
10
-8
and did
coelute
MR4 is eluted.
MR4 (at
and this
(Fig.3c)
which
not
Collectively,
order
= 5 (at
Thus,
whereas
10
from
via
aldosterone
-8
wanting
a target these
of MR4, aldosterone
serum
its
10 -7 M) was saturated
was totally is
with
aldosterone
= 1, R-5020
shown),
M) in the
not
in displacing
(Fig.lc),
of MR (5).
saturation (at
progesterone
= 0, progesterone
and is devoid
action
vs 3c),
14C
(T) where
studies
aldosterone
corticoids
(MR,)
potent
= 1, R-5020
(Fig.3a
O-0
C-corticosterone
in competition
the order:
tagen
14
bound
receptor
3H;
for
data
in
M Progesin
the
mineralo-
suggest
proges-
MR2, and a common locus = 2, progesterone
= 1 =
R-5020). Molecular
filtration
000 and 67,000 (Fig.4a) the
followed
other
hand,
renal
(Fig.4b)
dent
only
of
daltons,
by a broad bound
receptors
respectively) free
progesterone
and myocardial
in the kidney.
(D) was 50 times
steroid
With
more abundant,
revealed
of bound peak
(since
revealed
R-5020
in place
with
A and B in renal
with
an additional
(Fig.4c)
81
aldosterone untreated
cytosols
C was wanting,
peaks
peak
another
(122,
cytosol
charcoal).
On
(C) in both peak
(D) evi-
of progesterone,
this
peak
and A & B were
comparable
Vol.
89,
No.
1, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
b
0 FRACTION FRACTION
NUMBER
NUMBER
d
FRACTION
Fig.
NUMBER
FRACTION
4. MOLECULAR FILTRATION
NUMBER
OF STEROID BINDERS ON ULTROGEL ACA-44
RESIN
2 ml renal cytosol was incubated with 10W7M of either 3H-aldosterone (b), or 3H-R-5020 (d); 2 ml myocardium cytosol with '"27 3H-progesterone 10 ogesterone was used for Fig.4c; for Fig.4b, I ml serum with 0.25 UCi 0': " C-corticosterone was utilised. Except for 4a, charcoal treatment was used in all cases for better resolution of peaks. Equilibration and elutionwere carried out with 0.01 M phosphate + 0.1 M NaCl on columns of 1 x 130 cm,as previously described in detail (1,2,5). --------------A
280;
(Fig.4d).
The last
-
3H;
peak
in Figs.4b
dissociated
during
and B (Fig.
4b) and would
for
the
genesis
of
On Sephadex peak
D (Fig.5b).
only
(Fig.5c); Again,
of the
argue
the steroid
with
(C,D), based
relative
both
(aldosterone
cytosol
complex
of
in both
peaks
A
factor (1).
and charge, (Fig.
kidney
coeluted
the various
that
rechromatography weights
vs progesterone
82
after
upon molecular
14 C-progesterone
abundance
with
as the
as explained
3 H-progesterone
serum bound
coeluted
polymerisation
aldosterone-renal
seen with
to radioactivity
progesterone
against
peaks
14C
and 4c corresponds
Serum bound
columns,
A, C, and D were
and the heart
function
these
A-25
B was evident
peaks
elution.
-
only
components
in the kidney)
5a); (Fig.
5b)
with
peak
was a as well
Vol.
89,
No.
1, 1979
BIOCHEMICAL
FRACTION
AND
BIOPHYSICAL
RESEARCH
NUMBER
FRACTION
FRACTION
COMMUNICATIONS
NUMBER
NUMBER
Fig.
5. SEPARATION OF STEROID BINDERS ON SEPHADEX A-25 COLUMNS 3H-aldosterone 4 ml renal cytosol + 10s7M of either (a) or 3H-progesterone (b), or 4 ml cardiac cytosol + 10s7 M 3H-progesterone (c) were charcoal treated prilsr to chromatography on 1.6 x 90 cm columns equilibrated with 0. 01 M Tris-HCl, pH 7.5 and eluted with a gradient between 0 and 1 M KC1 in this buffer. For Fig. 5b, 2 ml serm + 0.5 nCi 14C-corticosterone was used (1,2,5 ). --------------A
as the
nature
evident
with
but
not
0-0
of the
tissue
(3,4),
receptor R-5020
in renal
Indeed,
MR4 that
was absent
from
distinct
from
MR2 bound
of these
molecules
mineralocorticoid denominated
huge quantities
non targets
agonist,
also
of elution)
progesterone,
has never
been
studied
>>> progesterone serum),
were
and that
deoxycorticosterone, in the
(7),
in this bound
to
was clearly concentrations a natural
same position
in the kidney
83
as also
its
with
when physiological
Since eluted
from
here
(liver,
aldosterone,
employed.
MR3 in order
which
of R-5020
were
of aldosterone
wasconfirmed
gestagen)
vs the heart),
above.
displacement
cytosol
context.
renal
in the kidney
chromatography
competitive
(an "ideal"
H;
(progesterone
Ultrogel-ACA-44
As before specific
3
280;
(previously but
not
the
liver,
Vol.
89,
No.
1, 1979
progestagens tin-like
may possess
separation the
Although
establishes interpretation kidney
the possibility presented
(8).
AND
BIOPHYSICAL
weak mineralocorticoid
MR4 may be the
autoradiography
for
BIOCHEMICAL
equivalent
that
between
mere
Scatchard
of receptor
activity
may be endowed of receptor
in our Multipolar
activity
of the unusual
The discordance
with
high
maturation model
RESEARCH
binder
competition type
of
COMMUNICATIONS
(3);
the
transcor-
observed studies
analyses
in kidney vs physical
are not
adequate
and may even be misleading. specific
from
progesterone
an uncommitted
receptors, protein
has been
(1,2).
ACKNOWLEDGEMENTS: Supported by grants from the INSERM, the DGRST (BFM 76 7 0725) and the CNRS (AI 03 1917). Thanks are due to Ms. M. Philippe for assistance and illustrations. REFERENCES 1. Agarwal, M. K. (1977), Multiple Molecular Forms of Steroid Hormone Receptors, pp 93-112, Elsevier/North Holland, Amsterdam. 2. Agarwal, M. K. (1979), Antihormones, pp 51-110, Elsevier/North Holland. 3. Landau, R. L. (1979) in "Antihormones" (M. K. Agarwal, Ed), pp 153166, Elsevier/North Holland, Amsterdam. 4. Wambach, G. and Higgins J. R. (1979), in "Antihormones" (M. K. Agarwal, Ed), pp 167-180, Elsevier/North Holland, Amsterdam. 5. Agarwal, M. K. (1975), Nature (Land.), 254, 623-625.. 6. Horowitz, K. B. and McGuire, W. L. (1975), Steroids, 26: 785-791. 7. Agarwal, M. K. (1976), FEBS Letters, 67, 260-263. 8. Strum, J. M., Feldman, D. and Edelman, I. S. (1976), Endocrinology, 2, 505-511.
84
89,
July
12, 1979
No.
1, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
COMMUNICATIONS
Pages
PARADOXICAL NATURE OF MINERALOCORTICOID +M. K. Agarwal
May 25,
77-84
RECEPTOR ANTAGONISM BY PROGESTINS
and
++
+CNRS and ++ UER Broussais-Hztel Received
RESEARCH
J. Paillard Dieu,
Paris,
France
1979
SUMMARY: Progesterone, but not the synthetic progestagen R-5020, was as good as aldosterone in displacing the mineralocorticoid from its specific receptor in rat kidney both in classical competition studies and in aldosterone binding to MRl and MR2 components of MR during physical separation. Paradoxically, at equimolar (10-S M) concentrations, both gestagens (progesterone = 1, R-5020 = 20) were preferentially bound to the MR4 component which coeluted with serum bound 14C-corticosterone (or 14C-progesterone), which could not be labelled with aldosterone in the kidney, and which could not be detgcted in the liver and the serum under any condition. The MRl entity, at loM, wa:s saturated as: aldosterone = 2, R-5020 = 1 = progesterone. Thus, the agonist and the antagonist function in mineralocorticoid action may be expressed by two or more distinct and different sites. INTRODUCTION: their
target
to nuclear this
specific
bits agoist
only
or via
mediated
binding
in competition
vector,
receptor
we have
corticoids for
an "ideal" recently
Abbreviations glucocorticoid
five
synthetic
of the
either
cellular
vector
(2).
diuresis
(3),
and antagonises
(4).
Contrary
polymorphism separation
of steroid (6),
R-5020
tritiated
form,
by an antihormone that
does not
MR = mineralocorticoid receptor.
disease,
of receptor
for
and this (1).
may proceed bind
of a
mineralo-
seems true
In the
(promegestone was used
inhireceptor-
hypothesis
hormones
complex
Proges-
to the
(5),
to
by reversing
Addison's
progestin in the
may act
to bind
with
physical
in the kidney
used are: specific
translocation
another
studies
classes
appear
in patients
demonstrated
antagonism
subpopulation
with
natriuretic
synthesized
mineralocorticoid that
all
subsequent
effects
during
hormones
'Antihormones'
association
aldosterone
corticosteroid
with
(1).
aldosterone
and gluco-
study,
sites
opposes
unitary
the
step,
receptor
acceptor
sequence
terone
In an initial
present 17-a--methyl),
to demonstrate via
of
that
saturation
of
aldosterone.
specific
receptor;
+Address after 15 July 1979: 15 Rue de 1'Ecole de MGdecine, 75270 Cedex 06, France, to whom all correspondance should be sent.
GR = Paris
0006-291X/79/130077-08$01.00/0
77
Copright All rights
@ 1979 by Academic Prrss. Inc. in unyform resrrved.
of reproduction
Vol.
89,
No.
BIOCHEMICAL
1, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
MATERIALS AND METHODS: Male, Wistar rats (150-200 g) were bilaterally adread libitum nalectomised 2-3 days prior to use; food and 1% NaCl were provided after surgery. Animals were sacrificed under ether anaesthesia, the desired organ was perfused with the initial buffer, according to the experimental procedure, and cell sap was finally obtained by centrifugation at 105,000 g (50 min). Blood, obtained by aortic cannulation was allowed to clot at 37°C (30 min), then at 4°C (60-90 min), and finally centrifuged at 3000 g. For competition and association studies, 0.5 ml cytosol (in 0.01 M Tris(60 min 4'C) in presence of a desired HCl, pH 7.4), or serum, was incubated amount of the radioactive steroid alone or in presence of a known excess of the cold homologous or heterologous molecule. Free steroid was removed by the addition of 0.5 ml (50 mg/ml) of activated charcoal (Sigma C-5260), further incubation (10 min 4"C), and centrifugation (15 min, 4000 g). Aliquots of 0.5 ml were mixed with 10 ml Scintix (Isotec, France) and counted in a Packard Tricarb Scintillation Spectrometer (1,2,5). Protein was quantitated by the Biuret method and all optical densities were read manually. organ cytosol, prepared in the initial buffer as per For chromatography, was equilibrated with the steroid and charcoal treated as elution procedure, above,and loaded onto the resin as described previously (1,2,5). Undiluted serum was similarly treated; further details are given in appropriate legends. 1,2,3H-aldosterone (53 Ci/m mol; lot 32), 1,2,6,7,3H-progesterone (95 Ci/ m mol; lot 26), 4- 14C-corticosterone (52 mCi/ m mol; lot 12), and 4-14Cprogesterone (61 mCi/m mol; lot 38) were purchased from Amersham (U.K.). 3HR-5020 (promegestone 17-a-methyl; 87 Ci/m mol; lot 1009-149) was a product of New England Nuclear (USA). Radiochemical purity exceeded 98% in all cases. RESULTS AND DISCUSSION:
Data
aldosterone
in both
targets
was similar of mineralocorticoid . to exhibit
appeared heart the
steroid; Data
and the to vectors
diluted
(l/10) in Fig.lc
displacing effective
establish
In a reciprocal kidney
bound
three
steroids
data
was far no binding
confirm
progesterone
regard, experiment
3H-R-5020. (aldosterone,
that from
its
(not In view
for
shown),
cold
progesterone,
78
(Fig.
whereas other
of receptor
in the
could
be of
R-5020
liver,
whereas
lb).
R-5020
synthetic
aldosterone multiplicity R-5020)
was far gestagens
very
poorly
(1,2,5) bind
of
(3,4).
was as good as aldosterone
receptor,
as reported
to the dose
Furthermore,
than
at all
the
characterstics
la vs lb).
greater
serum exhibited
in
of R-5020
association
(Fig.
binding
literature
quantities
of
two known
proportional
in the
greater
the
characterstics
bound
comparable
in the kidney
3H-aldosterone in this
was
progestin
of binding
progesterone
association
far
despite
"ideal"
However,
earlier
that
the kinetics
and the kidney,
(1).
different
confirm
cytosol,
binding
Data
mostly
in Fig.lb
aldosterone
heart
Serum aldosterone
these
to kidney
the
show that
hormones
somewhat
vs the kidney.
bound
in Fig.la
the
in less (3,4). displaced
, do these
same vector?
Vol. 89, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
t
STEROID
CONCENTRATION
(M)
Fig.
1. ASSOCIATION CHARACTERSTICS OF STEROID BINDING IN SEVERAL RAT TISSUES For Fig.la, renal cytosol was equilibrated with 3H-aldosterone (X) or 3Hprogesterone (A), myocardial with either 3H-aldosterone (0) or 3H-progesterone (a), and serum with 3H-aldosterone (X-----X). For Fig.lb, renal (X) or hepatic (C)) cytosol was incubated with 3H-R-5020. For Fig.lc, renal cytosol in presence of 10-8 M 3H-aldosterone was put in contact with the indicated concentration of non-radioactive aldosterone (O), progesterone (a), or R-5020 (X ). Non-specific binding in all cases was calculated by determination of radioactivity in presence of one thousandfold excess of cold, homologous steroid and was subtracted from the corresponding value obtained with tritiated hormone alone. Each sample (cold + radioactive or radioactive alone) was studied in triplicate at each point shown in the figure. For other details seeMethods and earlier publications (1,2,5).
Results MRl, Even
in Fig.2a
MR2 and GR type 10
-7
establish
of receptors
Pi progesterone
labelling
did
coeluted results
with
serum
transcortin
were
obtained
with
progesterone
in scale).
of
ralocorticoid
from
(3,4);
however,
logical
range,
than
its
action specific
at equimolar aldosterone
progestagen
progesterone
cytosol
to all
unlabelled
(Fig.2c)
progesterone
receptor
saturates
(please
components
can proceed
( 1o-8
M) binding
transcortin
via
in the
elution.
2a vs 2b).
bound
79
More
Similar
where
qunatitatively
note
the difference
in Fig.2a
diminished
(confirming
Fig.lc),
of the mine-
of progestagen
or 10 -7 M) concentrations,in a specific
the
to MR4 which
(Fig.Zb).
displacement region
to
MR2 whereas
(Fig. were
in the kidney
binding
higher
of aldosterone
aldosterone
myocardium
aldosterone 100 fold
with the
of
-8
aldosterone-specific
(T) bound
was bound
Since
in presence antagonism
of
(10
T = position
label
than
huge quantities
aldosterone
(5);
not
of MRl was greater
importantly,
less
further
MR2 and progeserone
the
excess physiothe MR4
Vol.
89,
No.
1, 1979
BIOCHEMICAL
GR
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
T
1
FRACTION
Fig.
FRACTION
NUMBER
NUMBER
FRACTION
NUMBER
2. ION EXCHANGE SEPARATION OF STEROID BINDERS ON DEAE-5'2 COLUMNS
5 ml renal cytosol was incubated with 10e8M 'H-aldosterone (a); 4 ml of either kidney (b) or myocardium (c) cytosol was incubated with 10e7M 3H-proin presence of 0.5 uCi of y;:E;z; ,E;zn;ig. *b 9 2 ml serum was incubated ; all samples were charcoal treated (100 mg/ml mixture), and loaded onto DE-52 columns (1 x 25 cm). separately, mixed for Fig.Zb, After a low ionic prewash (0.001 M phosphate, pH 7.5), elution was begun (at the arrow) with a gradient between 60 ml each of 0.001 M and 0.2 M of this buffer, pH 7.5. 1 ml samples were processed for radioactivity and the absorbance was recorded manually; further details in Methods and (1,2,5).
--------------A
which
is not
(see
below) Five
280;
0-0
transcortin and,
fold
first
second,
greater -7
3W
quantities
(10
reversed
MRl was considered
kidney
if bound
because
because
when R-5020
M) was used
R-5020
of
in place (Fig.
obtained
when undiluted
cytosol
and
was neither
excess
cold
coelutes too,
eluted
nonspecific
with
R-5020)
nor
MR4 (Fig.3a in the (displaced
coincided vs 3b).
same position by excess
14C
does not
competes
label
with
radioactivity
in the MR4 region
this
R-5020
R-5020
of radioactivity (Fig.3b)
0-0
are
Furthermore,
80
the
serum-R-5020 R-5020),
order
of
a small
peak
in place
of renal
in presence
14 C-corticosterone bound
was
quantities
with
(abolished
liver
MR4.
in the MR4 region
The tremendous
serum bound
of cold
for
to be contrasted
specific
as the
progesterone
but
serum was used
with
carrier
eluted
of progesterone, 3a vs 2b).
this
R-5020
of which
(Fig.3c),
complex
(Fig.3b),
was 50 times
less
was than
MR4
Vol.
89,
No.
1, 1979
BIOCHEMICAL
0-3)3Hfkh0-‘)
AND
EIOPHYSICAL
RESEARCH
COMMUNICATIONS
a
b
2
P ‘z -0 FRACTION
Fig.
NUMBER
FRACTION
NUMBER
3. ION EXCHANGE SEPARATION OF R-5020
FRACTION
NUMBER
BINDERS ON DEAE-CELLULOSE-52
4 ml renal cytosol (a), undiluted serum (b), or liver cytosol (c) was incubated with 10e7 M 3H-R-5020; 2 ml fresh serum with 0.5 PCi 14C-corticosterone was used for cochromatography as for Fig. 2 and (1,2,5). --------------A
-
280;
bound
R-5020
in the kidney
transcortin R-5020
was less
terone serum
than
(Fig.3b)
= 20,
and the
via
revealed
not liver
10
-8
and did
coelute
MR4 is eluted.
MR4 (at
and this
(Fig.3c)
which
not
Collectively,
order
= 5 (at
Thus,
whereas
10
from
via
aldosterone
-8
wanting
a target these
of MR4, aldosterone
serum
its
10 -7 M) was saturated
was totally is
with
aldosterone
= 1, R-5020
shown),
M) in the
not
in displacing
(Fig.lc),
of MR (5).
saturation (at
progesterone
= 0, progesterone
and is devoid
action
vs 3c),
14C
(T) where
studies
aldosterone
corticoids
(MR,)
potent
= 1, R-5020
(Fig.3a
O-0
C-corticosterone
in competition
the order:
tagen
14
bound
receptor
3H;
for
data
in
M Progesin
the
mineralo-
suggest
proges-
MR2, and a common locus = 2, progesterone
= 1 =
R-5020). Molecular
filtration
000 and 67,000 (Fig.4a) the
followed
other
hand,
renal
(Fig.4b)
dent
only
of
daltons,
by a broad bound
receptors
respectively) free
progesterone
and myocardial
in the kidney.
(D) was 50 times
steroid
With
more abundant,
revealed
of bound peak
(since
revealed
R-5020
in place
with
A and B in renal
with
an additional
(Fig.4c)
81
aldosterone untreated
cytosols
C was wanting,
peaks
peak
another
(122,
cytosol
charcoal).
On
(C) in both peak
(D) evi-
of progesterone,
this
peak
and A & B were
comparable
Vol.
89,
No.
1, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
b
0 FRACTION FRACTION
NUMBER
NUMBER
d
FRACTION
Fig.
NUMBER
FRACTION
4. MOLECULAR FILTRATION
NUMBER
OF STEROID BINDERS ON ULTROGEL ACA-44
RESIN
2 ml renal cytosol was incubated with 10W7M of either 3H-aldosterone (b), or 3H-R-5020 (d); 2 ml myocardium cytosol with '"27 3H-progesterone 10 ogesterone was used for Fig.4c; for Fig.4b, I ml serum with 0.25 UCi 0': " C-corticosterone was utilised. Except for 4a, charcoal treatment was used in all cases for better resolution of peaks. Equilibration and elutionwere carried out with 0.01 M phosphate + 0.1 M NaCl on columns of 1 x 130 cm,as previously described in detail (1,2,5). --------------A
280;
(Fig.4d).
The last
-
3H;
peak
in Figs.4b
dissociated
during
and B (Fig.
4b) and would
for
the
genesis
of
On Sephadex peak
D (Fig.5b).
only
(Fig.5c); Again,
of the
argue
the steroid
with
(C,D), based
relative
both
(aldosterone
cytosol
complex
of
in both
peaks
A
factor (1).
and charge, (Fig.
kidney
coeluted
the various
that
rechromatography weights
vs progesterone
82
after
upon molecular
14 C-progesterone
abundance
with
as the
as explained
3 H-progesterone
serum bound
coeluted
polymerisation
aldosterone-renal
seen with
to radioactivity
progesterone
against
peaks
14C
and 4c corresponds
Serum bound
columns,
A, C, and D were
and the heart
function
these
A-25
B was evident
peaks
elution.
-
only
components
in the kidney)
5a); (Fig.
5b)
with
peak
was a as well
Vol.
89,
No.
1, 1979
BIOCHEMICAL
FRACTION
AND
BIOPHYSICAL
RESEARCH
NUMBER
FRACTION
FRACTION
COMMUNICATIONS
NUMBER
NUMBER
Fig.
5. SEPARATION OF STEROID BINDERS ON SEPHADEX A-25 COLUMNS 3H-aldosterone 4 ml renal cytosol + 10s7M of either (a) or 3H-progesterone (b), or 4 ml cardiac cytosol + 10s7 M 3H-progesterone (c) were charcoal treated prilsr to chromatography on 1.6 x 90 cm columns equilibrated with 0. 01 M Tris-HCl, pH 7.5 and eluted with a gradient between 0 and 1 M KC1 in this buffer. For Fig. 5b, 2 ml serm + 0.5 nCi 14C-corticosterone was used (1,2,5 ). --------------A
as the
nature
evident
with
but
not
0-0
of the
tissue
(3,4),
receptor R-5020
in renal
Indeed,
MR4 that
was absent
from
distinct
from
MR2 bound
of these
molecules
mineralocorticoid denominated
huge quantities
non targets
agonist,
also
of elution)
progesterone,
has never
been
studied
>>> progesterone serum),
were
and that
deoxycorticosterone, in the
(7),
in this bound
to
was clearly concentrations a natural
same position
in the kidney
83
as also
its
with
when physiological
Since eluted
from
here
(liver,
aldosterone,
employed.
MR3 in order
which
of R-5020
were
of aldosterone
wasconfirmed
gestagen)
vs the heart),
above.
displacement
cytosol
context.
renal
in the kidney
chromatography
competitive
(an "ideal"
H;
(progesterone
Ultrogel-ACA-44
As before specific
3
280;
(previously but
not
the
liver,
Vol.
89,
No.
1, 1979
progestagens tin-like
may possess
separation the
Although
establishes interpretation kidney
the possibility presented
(8).
AND
BIOPHYSICAL
weak mineralocorticoid
MR4 may be the
autoradiography
for
BIOCHEMICAL
equivalent
that
between
mere
Scatchard
of receptor
activity
may be endowed of receptor
in our Multipolar
activity
of the unusual
The discordance
with
high
maturation model
RESEARCH
binder
competition type
of
COMMUNICATIONS
(3);
the
transcor-
observed studies
analyses
in kidney vs physical
are not
adequate
and may even be misleading. specific
from
progesterone
an uncommitted
receptors, protein
has been
(1,2).
ACKNOWLEDGEMENTS: Supported by grants from the INSERM, the DGRST (BFM 76 7 0725) and the CNRS (AI 03 1917). Thanks are due to Ms. M. Philippe for assistance and illustrations. REFERENCES 1. Agarwal, M. K. (1977), Multiple Molecular Forms of Steroid Hormone Receptors, pp 93-112, Elsevier/North Holland, Amsterdam. 2. Agarwal, M. K. (1979), Antihormones, pp 51-110, Elsevier/North Holland. 3. Landau, R. L. (1979) in "Antihormones" (M. K. Agarwal, Ed), pp 153166, Elsevier/North Holland, Amsterdam. 4. Wambach, G. and Higgins J. R. (1979), in "Antihormones" (M. K. Agarwal, Ed), pp 167-180, Elsevier/North Holland, Amsterdam. 5. Agarwal, M. K. (1975), Nature (Land.), 254, 623-625.. 6. Horowitz, K. B. and McGuire, W. L. (1975), Steroids, 26: 785-791. 7. Agarwal, M. K. (1976), FEBS Letters, 67, 260-263. 8. Strum, J. M., Feldman, D. and Edelman, I. S. (1976), Endocrinology, 2, 505-511.
84
