Vol. 275, Issue 2, H703-H709, August 1998
Hypothalamic angiotensin receptor subtypes in normotensive and
hypertensive rats
N. L.
Han and
M. K.
Sim
Department of Pharmacology, Faculty of Medicine, National
University of Singapore, Singapore 0511
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ABSTRACT |
The binding of
125I-labeled
[Sar1,Ile8]angiotensin
II to the hypothalamic membranes of the normotensive Wistar-Kyoto rat
(WKY) and the spontaneously hypertensive rat (SHR) was studied.
Displacement experiments with four centrally active angiotensins,
losartan, and PD-123319 confirm the known existence of angiotensin
AT1 and AT2 receptors in the rat
hypothalamus. The values of the inhibitory constants for angiotensin II
and PD-123319 in the SHR were significantly lower than the
corresponding values in the WKY, indicating the possible existence of
high-affinity hypothalamic AT1 and
AT2 receptors for the two ligands
in the SHR. The angiotensin AT1
receptor was further separated into a 5'-guanylyl
imidodiphosphate-sensitive and -nonsensitive subtype, indicating that
one of the subtypes is G protein coupled. The SHR has significantly
higher numbers of measurable
AT1-receptor subtypes as well as
AT2 receptor subtypes. The former
data support the findings of other investigators showing that the
hypothalamus of the SHR expressed more
AT1A and
AT1B mRNAs than that of the
normotensive rat.
Des-Asp1-angiotensin I, which is
known to attenuate the central pressor action of angiotensin II and
angiotensin III, acts on both the AT1 and
AT2 receptors, although it has a
higher affinity for the AT1
receptors. The overall increase in the number of
AT1 and
AT2 receptors in the SHR is in
line with the contention that the brain of the hypertensive rat,
compared with that of the WKY, has a hyperactive renin-angiotensin
system.
rat hypothalamus; des-aspartate-angiotensin I; angiotensin
AT1A receptor; angiotensin
AT1B receptor
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INTRODUCTION |
RECENTLY, WE HAVE SHOWN that
des-Asp1-angiotensin I, a
nine-amino acid peptide, attenuated the central action of angiotensin II in normotensive and hypertensive rats (31). Further study with the
use of D-amino acid-substituted
analogs of the nonapeptide demonstrated that
des-Asp1-angiotensin I is a
physiological peptide formed from angiotensin I by the action of a
specific aminopeptidase (21). The specific aminopeptidase has been
suggested to form a pathway for the degradation of angiotensin I that
bypasses the formation of angiotensin II (27). The activity of this
aminopeptidase in the hypothalamus of the spontaneously hypertensive
rat has been found to be significantly higher than in the corresponding
normotensive Wistar-Kyoto rat (30). These findings tend to indicate
that des-Asp1-angiotensin I is
involved in the central regulation of blood pressure and may have an
unknown role in hypertension. The present study characterized the
binding properties of the angiotensin receptor subtypes present in the
hypothalamus of the normotensive and hypertensive rat with the aim of
determining the receptor subtypes that likely mediate the central
action of des-Asp1-angiotensin I
in these animals.
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METHODS |
Animals and hypothalamic membranes.
Four-month-old male Wistar-Kyoto rats (WKY) and spontaneously
hypertensive rats (SHR) (280-310 g) were purchased from the Animal
Resource Centre. The systolic blood pressure values for the WKY and SHR
were 113 ± 14 and 165 ± 8 mmHg, respectively. The animals were
decapitated and their brains removed. The hypothalamus from each brain
was dissected out according to the procedure of Glowinski and Inversen
(5). Each hypothalamus of ~100 mg was homogenized in 10 vol of 50 mM
Tris · HCl (pH 7.4) using a Potter-Elvejhem homogenizer. The homogenate was centrifuged at 50,000 g for 10 min. The pellet was
resuspended in 10 vol 10 mM phosphate buffer (pH 7.4) containing (in
mM) 5 EDTA, 100 NaCl, and 0.1 phenylmethylsulfonyl fluoride (PMSF) and
was resedimented at 50,000 g for 5 min. This washing procedure was repeated once. The pellet was
resuspended in 400 vol of the same 10 mM phosphate buffer, which then
contained four additional compounds, namely, 0.2 mg/ml soybean trypsin
inhibitor, 0.018 mg/ml
o-phenanthroline, 2 mg/ml
heat-denatured bovine serum albumin, and 0.14 mg/ml bacitracin. The
procedure for the preparation of membranes was carried out under
identical conditions for both the WKY and SHR and was performed on the
same day. The binding protocol used was described by Chang et al. (4).
Binding experiments.
For saturation assay, 500-µl aliquots of membranes (containing an
equivalent of 1.25 mg wet wt of hypothalamus) were incubated at
37°C with increasing concentrations of
125I-labeled
[Sar1,Ile8]angiotensin
II (15-180 pM) in the absence and presence of unlabeled [Sar1,Ile8]angiotensin
II in a total volume of 550 µl. The relative concentration of
unlabeled
[Sar1,Ile8]angiotensin
II to
125I-[Sar1,Ile8]angiotensin
II was maintained at a constant ratio of 1,000:1. A similar protocol
was employed in a saturation assay using
125I-angiotensin II carried out in
the presence and absence of unlabeled angiotensin.
For displacement assay, 500-µl aliquots of membranes (containing an
equivalent of 1.25 mg wet wt of hypothalamus) were incubated at
37°C with a fixed concentration of
125I-[Sar1,Ile8]angiotensin
II (60 pM) and increasing concentrations (1 pM to 10 µM) of various
unlabeled ligands. For displacement carried out in the presence of
5'-guanylyl imidodiphosphate [Gpp(NH)p], the
concentrations of the guanosine nucleotide used were 1 µM, 50 µM,
and 1 mM.
The binding assays were terminated by centrifugation at 40,000 g for 1 h at 4°C. (Incubation of a
mixture of 60 pM
125I-[Sar1,Ile8]angiotensin
II and 1.25 mg hypothalamic membrane for 30, 45, 60, 75, and 90 min
showed that the binding reached equilibrium at 60 min.) The pellets
obtained were rapidly rinsed twice with 1 ml ice-cold
Tris · HCl buffer (pH 7.4) containing 0.15 M NaCl. The radioactivity trapped in the pellets was counted in a gamma counter
(Cobra Suto-Gamma 5002 Series) with an efficiency of 72%. The
integrity of
125I-[Sar1,Ile8]angiotensin
II after the 1-h incubation at 37°C was determined as follows. The
unbound
125I-[Sar1,Ile8]angiotensin
II in the supernatant of each initial displacement assay was separated
by HPLC, and the radioactivity was directly quantitated by a Beckman
radioisotope detector (model 710) as described previously (29). Ten
microliters of each supernatant were resolved through a Merck 5-µm
C18 column, and the
125I-[Sar1,Ile8]angiotensin
II was eluted by gradient chromatography (from 0.1% trifluoracetic
acid to 70% acetonitrile, 0.085% trifluoracetic acid). Controls show
that, under the incubation conditions, >90% of the radioligand
remained intact.
Data analysis.
The binding data were analyzed with the use of the computer programs
LIGAND (17) and EBDA (15) (both programs are Release 2.0, programmed by
A. Cuningham-Smith at BIOSOFT) to obtain the binding parameters given
in Tables 1-6.
Materials.
125I-[Sar1,Ile8]angiotensin
II and 125I-angiotensin II (2,200 Ci/mol) were purchased from NEN. Angiotensin II, angiotensin III, and des-Asp1-angiotensin I were
purchased from Bachem Feinchemikalein. Gpp(NH)p, PMSF, soybean trypsin
inhibitor, o-phenanthroline,
heat-denatured bovine serum albumin, bacitracin, and
[Sar1,Ile8]angiotensin
II were purchased from Sigma. Losartan and PD-123319 were gifts from
DuPont Merck Pharmaceutical and Parke-Davis, respectively.
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RESULTS |
Figure 1 shows the displacement of bound
125I-[Sar1,Ile8]angiotensin
II from the hypothalamic membranes of the WKY and SHR by its cold
equivalent and four other centrally active angiotensin peptides. The
inhibitory constant
(Ki) values are
given are given in Table 1. The
Ki values for
angiotensin II in the SHR were significantly lower than those in
the WKY. Figure 2 shows the displacement of bound
125I-[Sar1,Ile8]angiotensin
II from the hypothalamic membranes of the WKY and SHR by losartan and
PD-123319. The displacements were biphasic for both the antagonists,
with the inflection occurring at a concentration of
10
6 M of the antagonists.
This probably indicates the nonspecific displacement of the radioligand
from the AT1 and
AT2 receptor by PD-123319 and
losartan, respectively. The displacement of
125I-[Sar1,Ile8]angiotensin
II from the high-affinity binding sites by PD-123319 was significantly
greater in the SHR than in the WKY. Table 2 summarizes the Ki
values for the two antagonists. The
Ki value for the
specific PD-123319 binding site in the SHR was significantly lower than
that in the WKY.
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Fig. 1.
Displacement of 125I-labeled
[Sar1,Ile8]angiotensin
II from binding sites in hypothalamic membranes of Wistar-Kyoto rats
(WKY; A) and spontaneously
hypertensive rats (SHR; B) by
[Sar1,Ile8]angiotensin
II ( ), angiotensin II ( ), angiotensin III ( ),
angiotensin-(1 7) ( ), and
des-Asp1-angiotensin I ( ). Each
point on graph is mean ± SE of 5 experiments performed in
duplicate. Inhibitory constants for angiotensins are given in Table
1.
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Table 1.
Ki values of five angiotensin peptides in displacing the
binding of 125I-labeled
[Sar1,Ile8]angiotensin II from hypothalamic
membranes of WKY and SHR
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Fig. 2.
Displacement of
125I-[Sar1,Ile8]angiotensin
II from binding sites in hypothalamic membranes of WKY (open symbols)
and SHR (solid symbols) by losartan (squares) and PD-123319
(triangles). Each point on graph is mean ± SE of 5 experiments
performed in duplicate. * Significantly different from
corresponding values in WKY (P < 0.05, Student's t-test).
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Figure 3 shows the displacement plots for
specific
125I-[Sar1,Ile8]angiotensin
II binding to hypothalamic membranes of the WKY and SHR. The data
indicate the existence of two binding sites in the membranes of both
the animals. In the presence of 1 mM Gpp(NH)p, the biphasic Scatchard
plots were converted to linear plots that characterized only the
low-affinity binding sites. Table 3
summarizes the binding parameters. Figure 4
shows the Scatchard plots of 125I-angiotensin II binding to the
hypothalamic membranes of the WKY, and Table
4 summarizes the binding parameters. This
experiment was carried out to show that binding of both the antagonist
(e.g., 125I-[Sar1,Ile8]angiotensin
II) and agonist (e.g.,
125I-angiotensin II) to G
protein-coupled receptor was inhibited by guanine nucleotide as has
been reported by Avissar and Sokolovsky (1) but not by Harden et al.
(6).
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Fig. 3.
Scatchard plots of specific
125I-[Sar1,Ile8]angiotensin
II binding to hypothalamic membranes of WKY
(A) and SHR
(B) in absence () and presence
() of 1 mM 5'-guanylyl imidodiphosphate
[Gpp(NH)p]. B/F, ratio of bound to free
radioligand.
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Table 3.
Binding constants for 125I-labeled
[Sar1,Ile8]angiotensin II binding to
hypothalamic membranes of WKY and SHR
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Fig. 4.
Scatchard plots of specific
125I-labeled angiotensin II
binding to hypothalamic membranes of WKY in absence () and presence
() of 1 mM Gpp(NH)p.
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Figure 5 shows the Scatchard plots of the
saturation assays carried out in the presence of 1 mM PD-123319 and
losartan, respectively. When both the saturation assays were repeated
in the presence of 1 mM Gpp(NH)p, the Scatchard plots of the former
(i.e., in the presence of PD-123319) were converted to a linear plot
that characterized the low-affinity binding sites. The binding
parameters are summarized in Table 5.
Figure 6 shows the displacement of 125I-[Sar1,Ile8]angiotensin
II by des-Asp1-angiotensin I from
the hypothalamic membranes of the WKY and SHR in the presence and
absence of 1 µM losartan and PD-123319, respectively. The binding
parameters are summarized in Table 6.
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Fig. 5.
Scatchard plots of specific
125I-[Sar1,Ile8]angiotensin
II binding to hypothalamic membranes of WKY in presence of either 1 µM PD-123319 (A) or 1 µM
losartan (B) with presence () and
absence () of 1 mM Gpp(NH)p.
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Table 5.
Binding constants for 125I-labeled
[Sar1,Ile8]angiotensin II binding to
hypothalamic membranes of WKY and SHR in presence of losartan or
PD-123319
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Fig. 6.
Displacement of 125I-[Sar1,Ile8]angiotensin
II from binding sites in hypothalamic membranes of WKY
(A) and SHR
(B) by
des-Asp1-angiotensin I in absence
(circles) and presence of 1 µM losartan (squares) or 1 µM PD-123319
(triangles). Each point on graph is mean ± SE of 4 experiments
performed in duplicate.
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Table 6.
Ki values of des-Asp1-angiotensin I in
displacing the binding of 125I-labeled
[Sar1,Ile8]angiotensin II from hypothalamic
membranes of WKY and SHR in presence of losartan or PD-123319
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DISCUSSION |
The binding of
125I-[Sar1,Ile8]angiotensin
II to hypothalamic membranes of the WKY and SHR was displaced by four
angiotensin peptides, losartan, and PD-123319. This is in agreement
with the known existence of angiotensin
AT1 and
AT2 receptors in the rat
hypothalamus (11, 18, 39). The proportion of displacement of the bound
radioligand by the AT1 and
AT2 antagonists of 60:40 in the
WKY approximates the values of 60:40 and 75:15 reported by earlier
investigators in the normotensive Sprague-Dawley and Wistar rats,
respectively (11, 18). In addition, the displacement pattern (i.e.,
inflection from the high- to the low-affinity binding sites occurring
at concentrations of ~106
M of the antagonists) and the higher affinity of the
AT1 antagonist for its receptor
compared with that of the AT2
antagonist are also similar to the earlier findings (11, 18). Except
for angiotensin II and PD-123319, the
Ki values of the
ligands in the SHR were not significantly different from those in the
WKY (see Tables 1 and 2). This could indicate that the
AT1 and
AT2 receptors in the SHR exhibit
greater affinity for angiotensin II and PD-123319. PD-123319
(106 M) displaced ~37 and
50% of the total bound radioligand in the WKY and SHR, respectively
(see Fig. 2). Similarly, the percentages of
AT2 receptor obtained from the
saturation experiments carried out in the presence of
106 M losartan were 40 and
49% for the WKY and SHR, respectively (see Fig. 5 and Table 5).
However, the dissociation constant (Kd) of the
AT2 receptor for the radioligand
(see Table 5), although higher for the WKY (1.92 ± 0.69 × 109 M), was not
significantly different from that of the SHR (1.61 ± 0.34 × 109 M). These data,
together with those shown in Table 5, demonstrate for the first time a
significant increase in the measurable numbers of the
AT2 and
AT1 receptors in the hypothalamus
of the SHR.
The data obtained from saturation experiments carried out in the
absence and presence of Gpp(NH)p (see Fig. 3) divide the angiotensin
receptors into G protein-coupled and non-G protein-coupled subtypes.
Although the angiotensin AT1
receptors interact with G protein whereas the
AT2 receptors do not (3), it is
unlikely that the G protein-coupled and -noncoupled receptors
deciphered in this study correspond to the angiotensin
AT1 and
AT2 receptors, respectively. This
is due to the fact that the percentages of G protein-coupled receptor
subtype (29.8 and 26.7% for the WKY and SHR, respectively; see Table
3) and non-G protein-coupled subtype (75.3 and 79.6% for the WKY and
SHR, respectively; see Table 3) do not correspond to the percentages of
displacement by losartan (60 and 65% for the WKY and SHR,
respectively, see Fig. 2) and PD-123319 (37 and 50% for the WKY and
SHR, respectively, see Fig. 2). The reason became obvious when it was
found that the AT1 receptor can be
further separated into a G protein-coupled and -noncoupled subtype (see
Fig. 5). Hence, the
Kd and maximum binding numbers (Bmax) for the
high-affinity binding site given in Table 3 and Table 4 pertain to the
same G protein-coupled AT1
subtype, and the
Kd and
Bmax for the low-affinity binding site, obtained in the presence and absence of 1 mM Gpp(NH)p (given in
Table 3), pertain to a mixture of two receptor subtypes, the AT2 and the non-G protein-coupled
AT1 subtypes.
Recent cloning and expression studies have shown that the
AT1 receptor exists as two
subtypes, classified as AT1A and
AT1B (9, 10). Similarly,
functional study has also shown the existence in the rabbit pulmonary
artery of two isoforms of the AT1
receptor that are identifiable with the
AT1A and
AT1B subtypes (26). The
AT1B subtype present in the
cardiac portion of the pulmonary artery is G protein coupled and
indomethacin sensitive (26), whereas the
AT1A subtype present in the
pulmonary portion of the artery is not G protein coupled (28). This may
tie in well with the fact that the
AT1 receptor is known to
1) be directly coupled to
phospholipase C
1 (13), which generates inositol triphosphate independent of G protein, and 2)
stimulate the production of cAMP indirectly through
PGE2 production via a GTP binding
protein (36). Thus it is likely that the hypothalamic G protein- and
non-G protein-coupled AT1-receptor
subtypes are isoforms similar to the pulmonary receptor subtypes.
The values of Bmax given in Table
3 and Table 5 indicate that the hypothalamus of the SHR, compared with
that of the WKY, exhibits an increase in measurable numbers of the
AT1- and
AT2-receptor subtypes. Similarly,
the hypothalamus of the SHR, compared with that of the WKY, has also
been found by Raizada et al. (22) to express more
AT1A and
AT1B mRNAs. The present findings
support the contention that the brain of the SHR, compared with that of the WKY, has a hyperactive renin-angiotensin system as demonstrated by
higher angiotensin II levels (19), renin activity (7), angiotensinogen
mRNA (42), and greater response to intracerebrovascular angiotensin II
and angiotensin III (8, 31, 32, 40). Because the renin-angiotensin
system has been shown to be involved in blood pressure and volume
homeostasis via areas in the hypothalamus and brain stem (16, 20), the
increase in hypothalamic AT1 and
AT2 receptors lends credence to
earlier suggestions that this system also maintains the elevated blood
pressure in genetic forms of hypertension such as the SHR (19, 33, 35).
Hypothalamic angiotensin receptors may mediate hypertensive responses
such as an increase in water drinking, sodium appetite, sympathetic vasoconstriction, and vasopressin secretion. (14). For the latter response, Schiavone and co-workers (25) reported that angiotensin II-induced vasopressin secretion from rat hypothalamoneurohypophysial explants were inhibited by PD-123177 and CGP-42112A, whereas losartan was ineffective. Although direct functional evidence for a hyperactive renin-angiotensin system in the SHR is not available, recent cellular studies (2, 23) showed that hypothalamic nuclei of the SHR were more
sensitive than those of the WKY to angiotensin II-induced production of
early response gene products. The question of whether a hyperactive
central renin-angiotensin system present in the SHR would lead to
downregulation of cell surface angiotensin receptors remains
intriguing. Two recent studies on the cross talk between the adrenergic
and angiotensin receptors in the neurons of the SHR and WKY may provide
some insight. Lu and Raizada (12) have shown that the angiotensin
II-induced norepinephrine uptake in neuronal cultures is mediated by
the AT1 receptor, is significantly greater in the SHR than in the WKY, and is associated with parallel stimulation of mRNAs for c-fos and
norepinephrine transporter. Yang and co-workers (41) have shown that
treatment with 100 µmol/l norepinephrine for 8 h causes a 66%
downregulation of the AT1
receptors, an 83% decrease in
AT1-receptor mRNA, and a
significant attenuation of the
AT1-receptor-mediated stimulation
of norepinephrine transporter mRNA in neuronal cultures of the WKY but
not in those of the SHR. The absence of the cross talk between the two
receptors in the SHR brain may suggest that brain angiotensin receptors of the SHR are different.
Des-Asp1-angiotensin I acts on
both the angiotensin AT1 and
AT2 receptors (Fig. 6). It
antagonizes the central pressor action of angiotensin II and
angiotensin III (31, 32); hence, its efficacy and selectivity for the
receptor subtypes are different from those of angiotensin II and
angiotensin III. In the pulmonary artery,
des-Asp1-angiotensin I acts on
indomethacin-sensitive and -nonsensitive AT1-receptor subtypes to produce
opposite effects (26). With regard to the displacement of the
radioligand by
des-Asp1-angiotensin I in the
presence of losartan or PD-123319, the
Ki value for the
AT2 receptor is more than 10-fold
greater than that for the AT1
receptor, and this may indicate that the
AT1 receptors are preferentially
involved in the central action of
des-Asp1-angiotensin I. However,
findings from some laboratories (34, 37, 38) have shown that the
central AT1-receptor-mediated responses could be enhanced by blockade of the
AT2 receptor, suggesting that the
AT2 receptors exert a modulatory
effect on the AT1 receptors. The
involvement of angiotensin AT1
receptor subtypes in the central action of
des-Asp1-angiotensin I and other
angiotensins will remain an intriguing and challenging area for further
investigation. The advent of specific
AT1A and
AT1B agonists or antagonists will
be a breakthrough similar to the case with angiotensin-(17) in which
a specific antagonist of the heptapeptide, A-779, established
unequivocally that angiotensin-(17) acts on a new angiotensin
receptor (24).
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ACKNOWLEDGEMENTS |
The authors thank DuPont Merck Pharmaceutical and Parke-Davis for
the gifts of losartan and PD-123319, respectively.
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FOOTNOTES |
This study was supported by Grant RP-6600003 from the National
University Medical Institutes, Singapore.
Address for reprint requests: M. K. Sim, Dept. of Pharmacology, Faculty
of Medicine, National University of Singapore, Singapore 0511.
Received 9 December 1997; accepted in final form 10 April 1998.
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Abstract
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