245 s
Clinical Science (1981) 61,245s-247s
Specific nuclear binding of angiotensin I1
by rat liver and spleen nuclei
R. N. RE, A. A. M A C P H E E
AND
J. T. F A L L O N
Allon Ochsner Medical Foundation. New Orleans, LA, U.S.A.
Summary
1. Nuclei were isolated from rat liver and
spleen by a standard technique which preserved
the nuclear membrane. Electron microscopy,
DNA/protein ratios and S'-nucleotidase determinations codfirmed the purity of the preparation.
2. Both liver and spleen nuclei specifically
bound '251-labelled angiotensin I1 (ANG 11) with
high affinity ( K d Z 1-0 nmol/l). Saralasin ANG
I11 and ANG I1 competed with tracer for ligand
whereas ANG I did so less effectively and
neurotensin not at all.
3. These results suggest the presence of
specific nuclear intracellular ANG I1 receptors
and raise important questions regarding the
mechanism of action of this peptide.
Key words: angiotensin, nuclear binding.
Introduction
The peptide hormone, angiotensin I1 (ANG II), is
known .to bind specifically to target cell membrane receptors and thereby initiate at least some
of its physiological action [ 1-41. There is
evidence that many peptide hormones can gain
entry to cells [5-71, and there are data suggesting
that tritiated ANG I1 can localize in the nuclear
and mitochondria1 regions of myocardial and
smooth muscle cells [8-101. This finding has
raised the possibility that some 'late' actions of
the hormones, such as cellular hypertrophy or
protein synthesis, may be regulated by the
binding of the peptide to intracellular sites.
In order to investigate the possibility of
intracellular ANG I1 receptors more directly, we
isolated nuclei from the liver and spleens of male
Correspondence: Dr Richard Re, Alton Ochsner
Medical Foundation, 15 16 Jefferson Highway, New
Orleans, LA, U.S.A.
Wistar-Kyoto (WKY) rats and studied the ability
of 1251-labelledANG 11 to bind reversibly and
specifically to those nuclei.
Materials and methods
Nuclei were purified from the livers and spleens of
male WKY rats by modification of the method of
Aldridge [ 11I.
Binding studies were performed in one of two
ways. The first method employed 12sI-labelled
ANG I1 (New England Nuclear Corp., Boston,
MA, U.S.A.; 1000-1500 mCi/mg) at concentrations from 0.1 to 5.0 nmol/l. 1251-labelled
ANG I1 was incubated with 100 pl of isolated
nuclei for 0.5 to 120 m i n k Beckman Microfuge
tubes. All determinacmns were in duplicate or
triplicate and for each binding point a comparable set of tubes was incubated in the presence
mol/l) ANG 11.
of nuclei, tracer and excess (
After incubation at 22OC, tubes were spun and
aliquots of supernatants were removed. Both the
supernatant and the remaining pellet were then
counted for radioactivity and bound counts
calculated. Specific binding was defined as the
difference between the binding in the presence and
absence of ANG I1 at
mol/l.
In a second series of experiments, 1251-labelled
ANG I1 ( 0 . 1 4 . 3 nmol/l) was incubated in the
presence of 5-isoleucine ANG I1 (Sigma Corporation) at concentrations ranging from 0.5 to
5.0 nmol/l. Once again a Microfuge binding
system was employed as described above, and
specific binding was defined as the difference in
tracer binding obtained in the presence or absence
of ANG I1 at
mol/l.
Electron microscopy was performed on nuclear
preparations by standard techniques [121.
Protein was measured by a modification of the
method of Lowry [131 and DNA by a modification of the method of Burton [141.
R . N . Re, A . A . MacPhee and J. T. Fallon
246 s
5'-Nucleotidase activity was determined by a
modification of the method described by Goldfine
et al. [151.
In addition, displacement studies were performed by incubating tracer 'ZSI-labelledANG I1
with various concentrations of [des-Aspl-angiotensin I1 (ANG II), saralasin, ANG I and
neurotensin. For each binding point a series of
tubes incubated in the presence of ANG I1 at
mol/l was employed to determine nonspecific binding.
Finally, cell membranes were prepared by a
modification of the method of Ray [ 161.
Results
Transmission electron microscopy revealed that
the nuclear preparations studied consisted of
undamaged nuclei free of significant cell membrane or cytoplasmic contamination. Nuclear
membranes were not removed by the isolation
procedure.
ProteidDNA ratio for spleen nuclei was 2.3 ?
0.4 and for liver nuclei 3.2 f 0.2. These ratios
were consistent with the ratios expected for pure
lymphocytes or hepatocytes respectively. 5'Nucleotidase activity of the nuclear preparations
was less than 2% of that of the intact cells, again
indicating that the preparations were free of
significant membrane contamination.
Studies employing both spleen and liver nuclei
incubated in the presence of 12sI-labelledANG I1
revealed high affinity saturable binding to both
liver and spleen nuclei. Time course studies
revealed that the binding to liver nuclei was rapid,
with equilibrium being achieved in about 15 min.
Therefore binding studies were performed with an
incubation time of 25 min. Scatchard analysis
revealed a single class of binding sites with an
apparent affinity constant of 0.92 nmol/l in
spleen and 1-2nmol/l in liver nuclei.
In studies employing unlabelled ANG I1 in
concentrations ranging from 0.1 to 5.0 nmol/l for
displacement of tracer ANG 11, similar results
8
008
t
I
,
2 0
,
40
,
.
.
,
6 0 8 0 100120140160180
Bound ( f m o l h b e )
FIG.1. Scatchard analysis of angiotensin I1 binding to
isolated hepatic nuclei from the rat.
were obtained. An affinity constant for spleen
nuclei of 1.0 nmol/l was obtained and for hepatic
nuclei 1.1 nmol/l (Fig. 1).
Displacement studies revealed that ANG 11,
ANG I11 and saralasin were equally effective in
competing with 12sI-labelledANG I1 for nuclear
binding, whereas ANG I was only about onehundredth as effective and the unrelated peptide
neurotensin could not be shown to compete for
binding even at a
mol/l concentration.
Discussion
The competitive displacement studies which we
have performed indicated that the nuclear ANG
I1 receptor recognizes not only ANG 11, but also
ANG I11 and saralasin. As such, it has many
shared characteristics with other target cell
membrane receptors, such as those on the arterial
smooth muscle and the adrenal zona glomerulosa
cell. We detected some competition for ANG I1
binding by ANG I but this required concentrations of the decapeptide approximately 100
times greater than that of ANG I1 for comparable
levels of displacement. It also remains unclear
whether some of the ANG I-induced displacement resulted from conversion in the assay system
into ANG 11. The finding that the unrelated peptide, neurotensin, does not displace tracer ANG I1
from nuclei, further confirms the potential physiological significance of the receptor we have
identified.
Our contention that the binding we have
observed results from the existence of nuclear
binding sites is contingent upon the demonstration that the preparations employed were free
of membrane contamination. Electron microscopy revealed the preparations to be pure.
DNA/protein ratios were consistent with a pure
nuclear preparation free of significant membrane
contamination. 5'-Nucleotidase measurements
demonstrated that the nuclear preparations contained only a small fraction of the cellular content
of this cell membrane bound enzyme. Finally,
when in additional experiments hepatic and
splenic membranes were isolated specifically and
binding studies performed using these membrane
preparations, a different pattern of binding was
detected. Liver membranes were found to degrade
tracer ANG I1 rapidly at 22OC in the absence of
dithiothreitol. However, nuclear preparations did
not degrade tracer under these conditions and
definite binding could be detected in the absence
of dithiothreitol. The apparent affinity constant
for hepatic membranes in our system was about
0.32 nmol/l and this again suggested that the
hepatic membrane and nuclear sites were distinct.
Nuclear angiotensin.II binding
For spleen membrane it was quite difficult with
our techniques reliably to detect specific high
affinity ANG I1 binding. Although in some
experiments spleen membrane preparations could
be shown to bind ANG I1 specifically, we found
no evidence for sufficient quantities of high
affinity ANG I1 binding sites on spleen membrane to account by contamination for the
observed nuclear binding. Thus we have provided
multiple lines of evidence to exclude membrane
contamination as a cause for our results. Finally,
it is possible that the binding we have observed
results from the presence of a non-nuclear
intracellular receptor which migrates to the
nucleus during our isolation procedure. Although
our electron microscopy studies revealed no
evidence of such contamination of our nuclear
pellets, this possibility cannot be entirely
excluded.
The present study suggests the existence of
nuclear binding sites for ANG I1 and once again
raises the possibility of an intracellular site of
action for this hormone. The fact that we have
detected these receptors on the nuclei of cells not
usually considered to be ANG I1 target cells
additionally raises interesting questions about the
role of ANG I1 in cellular physiology.
Acknowledgments
Supported in part by National Heart, Lung, and
Blood Institute, Grant no. HL26101 and by
funds provided by the Alton Ochsner Medical
Foundation. We appreciate the technical assistance of Meera Parab and the editorial assistance
of the Medical Editorial Department of the Alton
Ochsner Medical Foundation.
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