Department of Biochemistry and Molecular Biology, Faculté de
Médecine de Paris-Ouest, Université René Descartes,
Paris, France
This study investigates the incidence of high-salt diet in blood
pressures, renal 
2-adrenoceptor subtypes distribution,
and gene expression in salt-sensitive (SBH) and salt-resistant (SBN) Sabra rats. Comparisons have been made between SBH and SBN rats submitted to a normal or a high-salt diet for 6 weeks. Only
2B-adrenoceptors are detected in kidneys of SBH rats,
whatever the diet. In contrast, mRNA corresponding to
2A- and 2B-subtypes are found in this substrain. In these rats, high-salt diet increases blood pressures and
up-regulates gene expression and density of only
2B-adrenoceptors. Inversely, 2A- and
2B-adrenoceptors and corresponding mRNA are found in
kidneys of SBN rats. In these rats, a high-salt diet does not affect
blood pressures but increases gene expression and densities of both
2A- and 2B-adrenoceptors. If the
up-regulation of renal 2B-adrenoceptor subtypes is
indicative of the hypertensive phenotype, the present study shows that
this mechanism is also present in normotensive salt-resistant Sabra
rats. In fact, the absence of 2A-adrenoceptors in SBH
could be responsible for the lack of adequate receptor-mediated renal
functions predisposing to salt-sensitivity and consequently the
development of hypertension. Conversely, the presence of this receptor
in SBN rats and its up-regulation could be protective change against
the increase of 2B-adrenoceptors induced by the salt
overload and could consequently be responsible for the resistance to
salt-induced hypertension.
 |
Introduction |
The
kidney plays a major role in the chronic regulation of blood pressure
via modulation of sodium and water excretion (Hall et al., 1990
;
Guyton, 1991). Several authors have focused on the role of the renal
2-adrenoceptors in the modulation of water clearance and sodium excretion (Strandhoy et al., 1982; Gellai and
Ruffolo, 1987), and a common characteristic among genetic models of
hypertension is an increase in renal
2-adrenoceptor density (Pettinger et al.,
1982; Parini et al., 1983, 1987). Because the physiologic activity of
2-adrenoceptors depends on their density
(Duzic et al., 1992), alteration in renal
2-adrenoceptor density could account for
disruption in blood pressure control.
Two different isoforms of 2-adrenoceptors have
been described in rodent kidney (Uhlen and Wikberg, 1991). The
2A-adrenoceptor plays a crucial role in the
hypotensive response (MacMillan et al., 1996) and is able to increase
urine flow rate solely by increasing renal osmolar clearance (Intengan
and Smyth, 1997a). On the other hand, the
2B-adrenoceptor subtype mediates the
2-adrenoceptor agonist-induced increase in
blood pressure (Link et al., 1996), increases the renal free water
clearance (Intengan and Smyth, 1996), and is overexpressed in the
kidney of spontaneously hypertensive rats (Gong et al., 1994, 1995).
The Sabra rat is a genetic model of salt-induced hypertension
(Ben-Ishay and Yagil, 1994). In contrast to the Sabra salt-resistant (SBN) rat, Sabra salt-sensitive (SBH) rats fed a regular diet, have
borderline hypertension at an early age, maintain a slightly elevated
blood pressure in adult life, and become invariably hypertensive in
response to relevant stimuli such as a deoxycorticosterone acetate-salt
or a high-sodium diet. On a normal diet, urinary flow and the excretion
of sodium, potassium, and total solutes are significantly lower in SBH
than in SBN rats. However, the urine osmolality of the SBH rat is
approximately 2-fold higher than that of the SBN rat. It has been
suggested that these findings in SBH rats are compatible with an
impaired renal excretion of sodium in the presence of a slight increase
in blood pressure (Ben-Ishay and Yagil, 1994). This peculiar feature in
the Sabra model is consistent with renal alteration in the distribution of 2-adrenoceptor subtypes. As a matter of
fact, SBH rats fed a regular diet, in contrast to SBN rats, exhibit
high-renal 2B-adrenoceptor densities and the
absence of the 2A-adrenoceptor subtype (Le Jossec et al., 1995). Recently, it has been shown that mice lacking one
copy of the 2B-adrenoceptor gene are unable to
develop salt-induced hypertension (Makaritsis et al., 1999a). These
data suggest the potential importance of
2B-adrenoceptors to control blood pressure in
response to dietary salt loading.
The aims of this study were to determine whether an hypertensive
stimulus such as high-salt diet alters the renal distribution of
2-adrenoceptor subtypes in Sabra rats and, if
so, to establish whether such alterations reflected changes in the
expression of their encoding genes.
| |
Experimental Procedures |
Animals.
We used original SBH and SBN male Sabra rats (aged
3 weeks) bred at the Center de Sélection et d'Elevage des
Animaux de Laboratoires (Orléans, France). Four groups of rats
were studied, two of each substrain: one with normal (0.2%) and one
with high (8%)-NaCl laboratory chow. Water was given ad libitum, and
rats were maintained at a constant room temperature (24°C) on a 12-h
light/dark cycle. After 6 weeks, systolic blood pressure was measured
between 9 and 11 AM using the tail-cuff method, with an
electrosphygmomanometer (Physiograph MK III; Narco-Bio-System, Inc.,
Houston, TX) on unanesthesized, restrained rats warmed to 38°C for 10 min. Two days later, rats were killed by decapitation, and their
kidneys were carefully removed and rapidly frozen in liquid nitrogen.
All experimental protocols were approved by the University Animal Use
and Care Committee.
Radioligand Binding Studies.
Renal membranes were prepared
from whole kidney and radioligand-binding studies performed, as
described previously (Le Jossec et al., 1995), with the specific
radiolabeled 2-adrenoceptor antagonist,
[3H]RX821002. Briefly, 300 µg of renal
membranes was incubated with 0.3 to 30 nM
[3H]RX821002, 1 mM EDTA-K, 100 µM
5'-guanylylimidodophosphate, 140 mM NaCl, and 50 mM Tris-HCl (pH 7.4),
in a final volume of 300 µl for 45 min at 25°C. Reactions were
stopped by dilution with ice-cold incubation buffer and rapid vacuum
filtration through Whatman GF/C filters (Whatman, Maidstone, UK). The
filters were washed twice with ice-cold incubation buffer and the
radioactivity retained on filters was quantified by liquid
scintillation counting. Nonspecific binding was determined in the
presence of 20 µM phentolamine and represented 10% of the total
binding. For competition studies, membranes were incubated for 45 min
at 25°C with 2 nM [3H]RX821002 (a
concentration near Kd value) and
either 0.1 nM to 1 mM guanfacine, a selective
2A-adrenoceptor agonist (Uhlen and Wikberg,
1991), or 0.1 nM to 1 mM prazosin, which is selective for the
2B-adrenoceptor (Bylund et al., 1988). The
resultant saturation and competition curves were analyzed using a
nonlinear least-squares curve fitting program (GraphPad PRISM; GraphPad Software, Inc., San Diego, CA). Protein concentrations were determined according to Bradford (1976) using 
-globulin as the standard.
Analysis of Renal 2-Adrenoceptor mRNA.
Total
renal RNA was isolated using guanidium thyocyanate-phenol-chloroform
extraction with the TRIzol reagent (Invitrogen, Cergy-Pontoise, France)
and used for RNA-directed complementary cDNA synthesis and DNA
amplification as previously described (Le Jossec et al., 1995), except
that HotStarTaq DNA polymerase (Qiagen S.A., Courtaboeuf, France) was
used according to the conditions provided by the supplier and that 1 µCi of [3H]dCTP was added to the PCR
reaction. PCR mixtures of cDNA and respective primers (Table
1) were amplified using a program
temperature control system (Appligene Oncor, Illkirch, France). One
cycle of PCR consisted of 1 min at 94°C, 1 min at 57°C, and 1 min
at 72°C for 2-adrenoceptor subtypes cDNA and
was performed for a total of 33 cycles. For 
-actin, similar
experimental conditions were used, except that the annealing
temperature was 53°C and the number of amplification cycles was 30. Primers used for -actin amplification (Nudel et al., 1983) were
chosen to span two introns to discriminate the cDNA amplification
products from genomic DNA contamination. Each reaction mixture was
separated on a 1.5% low melting point agarose (Invitrogen) gel stained
with ethidium bromide and documented on Polaroid 665 film (Polaroid UK,
Ltd., St. Albans, UK). For quantification, respective bands for
2A, 2B, and -actin signals were excised and agarose-melted at 70°C, and the incorporated radioactivity was determined by scintillation counting in Aquasafe 300 Plus (Zinsser Analytic, Frankfurt, Germany). The incorporated radioactivity was normalized with respect to the length of the three
cDNA, and 2A and 2B
messenger RNA levels were expressed versus -actin mRNA content.
Materials.
[3H]RX821002 (2.29 × 1012 Bq/mmol),
[3H]dCTP (1.92 × 1012 Bq/mmol), and DNA molecular weight markers
(100-bp ladder) were purchased from Amersham Pharmacia Biotech (Les
Ullis, France). Oligonucleotides were synthesized by Eurogentec
(Herstal, Belgium). The following drugs were supplied by the indicated
companies: prazosin (Pfizer Central Research, Sandwich, Kent, UK),
guanfacine (Novartis, Basel, Switzerland). All other materials were
obtained from Sigma Aldrich (Saint-Quentin-Fallavier, France).
Statistical Analysis.
All results were expressed as the
mean ± S.E.M. Statistical analyses were performed using analysis
of variance followed by the Student-Neuman-Keuls multiple comparison
test. Data from DNA amplification were analyzed using the nonparametric
one-way procedure of the SAS system (SAS Institute Inc., Cary, NC), and
comparison between groups were made using the
2 and Kolmogorov-Smirnov tests.
P < 0.05 was considered as statistically significant.
| |
Results |
Physiological Data.
At 6 weeks of regimen, blood pressures
were in normal diet (133 ± 9; 108 ± 10 mm Hg,
n = 6, P < 0.001 SBH versus SBN) and in sodium overload (161 ± 19; 112 ± 14 mm Hg,
n = 6, P < 0.005 SBH versus SBN). Body
weights were for SBH (238 ± 6; 234 ± 16 g, 0.2%
versus 8.0% NaCl) and for SBN (221 ± 7; 220 ± 13 g,
0.2% versus 8.0% NaCl).
Renal 2-Adrenoceptor Densities and Subtype
Distribution.
Specific binding of
[3H]RX821002 to kidney membranes isolated from
SBH and SBN rats was a saturable process (Fig.
1). Scatchard plots of
[3H]RX821002 binding were monophasic,
suggesting only one component of binding (data not shown). In all
experiments, Scatchard plots were best analyzed by a model with only
one high-affinity class of sites for both SBH and SBN rats and whatever
the diet. However, under normal diet, renal
2-adrenoceptor-binding capacities were significantly higher in SBH (169.7 ± 7.2 fmol/mg protein,
n = 6) than in SBN (116.8 ± 4.6 fmol/mg protein,
n = 6) (Fig. 2). Under
sodium overload, binding capacities were significantly increased in
both SBH (216.7 ± 8.5 fmol/mg protein, n = 6) and
SBN (165.4 ± 5.1 fmol/mg protein, n = 6) rats and
with no significant difference in the magnitude of this effect between
SBH and SBN. In addition, no differences in binding affinities were
observed between SBH and SBN and between normal and high-salt diet
(Table 2).
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Fig. 1.
Saturation curves of [3H]RX821002
specific binding to kidney membranes of SBH and SBN rats under normal
( ) and high-salt diet ( ). Data are representative of six separate
experiments performed in duplicate.
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Fig. 2.
Renal [3H]RX821002-binding capacities
of SBH and SBN under normal (open column) and high-salt diet (hatched
column). Results are expressed as mean ± S.E.M.
(n = 6) of binding capacities
(Bmax in fmol/mg protein) as described under
Experimental Procedures. °°°,
P < 0.001 for the comparison between SBH and SBN;
*, P < 0.05; ***, P < 0.001 for the comparison between normal and high-salt diet.
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TABLE 2
Binding characteristics of renal 2-adrenoceptors
Data shown are mean ± S.E.M. of six separate experiments. Each
experiment was conducted in duplicate.
Kd25°C, equilibrium dissociation
constant obtained from saturation curves (Fig. 1).
Ki, inhibition constants obtained from competition
experiments (Fig. 3). In parentheses, relative proportions of high
affinity sites for each competitor.
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Competition binding experiments were performed to discriminate the
2-adrenoceptor subtypes using prazosin
(selective for 2B-adrenoceptor) and guanfacine
(selective for 2A-adrenoceptor). In kidney
membranes of SBN rats after normal or high-salt diet, the competition
curves for prazosin and guanfacine were shallow (Fig.
3, bottom). Using the iterative
curve-fitting program, the curves were best fitted with the model of
two classes of sites. The analysis further shows that under normal
diet, the proportion of the high-affinity site for prazosin is 75% to
85% and the high-affinity site for guanfacine is 15% to 25%,
corresponding to the characteristics of 2B-
and 2A-adrenoceptor subtypes, respectively
(Table 2). From these results, the calculated
2A- and
2B-adrenoceptor densities in SBN represent,
19.5 ± 1.1 and 99.2 ± 5.4 fmol/mg protein, respectively. Interestingly, under high-salt diet, the proportion of
2A-adrenoceptor subtype reached 30% to 40%
of the total [3H]RX821002-binding capacity.
Therefore, the increase in 2-adrenoceptor density observed in SBN rats after high-salt loading appears due to
both enhanced 2A- and
2B-adrenoceptor subtype densities, 48.0 ± 1.6 and 132.8 ± 4.3 fmol/mg protein, respectively
(P < 0.05 n = 6). In contrast, in kidneys of SBH
rats under normal or high-salt diet, competition curves for prazosin
and guanfacine were steep and monophasic (Fig. 3, top). In all
experiments, the curves were best fitted with a one-site model.
Computer analysis of all curves indicated indeed that prazosin binds to
membranes with only high affinity and guanfacine with low affinity
(Table 2), thus showing that [3H]RX821002
binding sites have the characteristics of the
2B-adrenoceptor subtype. As a consequence, the
2A-adrenoceptor subtype was undetectable in
SBH rats and the increase in 2-adrenoceptor
density observed under high-salt diet in these rats resulted only from
a raise in the 2B-adrenoceptor subtype.
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Fig. 3.
Guanfacine (left) and prazosin (right) compete for
2-adrenoceptor [3H]RX821002 binding sites
on renal membranes of SBH rats (upper panels) and SBN rats
(bottom panels) under normal () and high-salt diet (). Data are
representative of six separate experiments performed in duplicate.
Total specific [3H]RX821002-binding capacities
(Bmax in fmol/mg protein) are extrapolated
according the relation Bmax = Bound
x(Kd 25°C + L)/L. Bound is the capacity observed in the
experiment in fmol/mg protein; Kd25°C is
the equilibrium dissociation constant for [3H]RX821002
obtained from saturation experiments (in nM); and L is
the concentration in radioligand in the experiment (in nM).
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Renal 2-Adrenoceptor Subtype Gene Expression.
Experiments using reverse transcription-polymerase chain reaction
(RT-PCR) were performed with specific
2-adrenoceptor subtype primers to determine
whether alterations in renal 2-adrenoceptor subtype distribution could be associated with modifications in mRNA
levels. cDNA amplifications with these specific primers show amplified
products of predicted size [311 bp for
2A-adrenoceptor (Fig.
4, upper panel) and 407 bp for
2B-adrenoceptor (Fig. 4, middle panel)] in
the kidney of SBH and SBN rats, whatever the salt diet. On the other
hand, the fragment generated from -actin primers (280 bp) (Fig. 4,
bottom panel), which is present at comparable levels in the four
groups, is the only fragment amplified, ruling out any genomic DNA
contamination (Fig. 4A). After normal salt diet, analysis of the
radioactivity incorporated in the
2-adrenoceptor products normalized to
-actin reveals (Fig. 5) higher
2B (P < 0.01) but equivalent
2A mRNA levels in SBH than in SBN rats. Under
high-salt diet (Fig. 5), the 2B-adrenoceptor
mRNA signal is increased in both SBH and SBN rats with a weaker
magnitude in SBH yielding thus to equivalent levels in SBH and SBN
rats. In contrast, the 2A-adrenoceptor signal
remains unchanged in SBH rats, whereas it is markedly increased in SBN.
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Fig. 4.
Ethidium bromide staining of RT-PCR products using
2-adrenoceptor subtypes or -actin primers in kidney
of SBH and SBN rats under normal and high-salt diet. Results are
representative of one experiment performed at 33 cycles of
amplifications for 2-primers and 30 cycles for
-actin. DNA size markers, lanes 1 and 6; SBH under normal (lane 2)
and high-salt diet (lane 3); SBN under normal (lane 4) and high-salt
diet (lane 5). The upper panel for 2A products, middle
panel for 2B products and bottom panel for -actin
products.
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Fig. 5.
Gene expression of 2-adrenoceptor
subtypes determined by RT-PCR normalized to corresponding -actin
levels and expressed as percent of SBH values under normal diet. Data
for normal (open column) and high-salt diet (hatched column) are given
as the mean ± S.E.M. (n = 6). °°,
P < 0.01 for the comparison between SBH and SBN;
*, P < 0.05; ***, P < 0.001 for the comparison between normal and high-salt diet.
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Discussion |
The aim of the present study was to determine whether elevation in
salt intake induces changes in renal
2-adrenoceptor subtype densities. To reach
this target we have compared Sabra rats differing in their blood
pressure response to high-salt diet. After 6 weeks of high-salt diet,
salt-sensitive Sabra rats exhibit significant increases in blood
pressures when compared with salt-resistant Sabra rats. In addition,
based on radioligand-binding studies, high-salt diet produces a
significant raise in 2-adrenoceptor densities
in kidneys of both SBH and SBN rats. Nevertheless, the total
2-adrenoceptor density remains significantly
higher in SBH than in SBN rats.
Renal 2-adrenoceptors are heterogeneous (Uhlen
and Wikberg, 1991), and altered distribution of their subtypes has been
considered to predispose the development of hypertension in Sabra rats
(Le Jossec et al., 1995). In fact, SBH rats fed a regular diet exhibit higher 2B-adrenoceptor densities in renal
cortex than SBN rats (Le Jossec et al., 1995). These high levels, which
appear to be the consequence of an overexpression of the encoding gene,
have also been observed in spontaneously hypertensive rats (Gong et al., 1994; 1995). Inasmuch as this overexpression is a common feature
of genetic models of hypertension, it has been hypothesized that the
2B-adrenoceptor overexpression could be
controlled by genetic factors predisposing the rat to the onset of
hypertension (Gong et al., 1994). A role for the
2B-adrenoceptor in the development of high
blood pressure is further strengthened by the following recent studies.
In 2B-adrenoceptor knockout mice, a lack of
immediate hypertensive response to
2-adrenoceptor agonists has been observed (Link et al., 1996). Moreover, mice lacking a full complement of the
2B-adrenoceptor gene are unable to raise blood
pressure in response to chronic salt loading after subtotal nephrectomy (Makaritsis et al., 1999a). In the present study, by the use of selective competitors, we find that high-salt diet increases renal 2B-adrenoceptor densities in both SBH and SBN
rats with marked elevation of blood pressure only in SBH. In addition,
our RT-PCR data indicate that these increases in
2B-adrenoceptor densities are secondary to the
overexpression of the gene encoding this receptor subtype. However,
after a high-salt diet, 2B-adrenoceptor densities are still higher in the SBH rat. Interestingly, we have recently reported a similar increase in renal
2B-adrenoceptor gene expression and density in
cafeteria-fed rats, a nongenetic model of obesity-related hypertension
(Coatmellec-Taglioni et al., 2000). Altogether, these observations lead
us to postulate that a high density in renal
2B-adrenoceptors could be a determinant factor
contributing in general to the hypertensive phenotype.
On the other hand, a role for the renal
2A-adrenoceptor subtype should also be
considered as it is also expressed in renal cortex of SBN rats to a
lesser extent than the 2B-subtype and is
pharmacologically absent in SBH rats under normal salt diet (Le Jossec
et al., 1995) as well as under high-salt diet as presently shown.
However, the 2A-adrenoceptor mRNA is clearly
present in the kidneys of SBH rats under both a normal and a high-salt
diet. These discrepant findings could be explained by the limited
sensitivity of the binding technique, which is unable to detect low
levels of 2A-adrenoceptor sites, if any, in
SBH rats. Another explanation could be that post-transcriptional or
post-translational events occur in the kidney of SBH rats preventing
detection of the receptor protein by binding studies. In contrast to
2B, 2A-adrenoceptor mRNA levels are completely insensitive to the high-salt diet in SBH
rats. Interestingly, a similar situation has been found in the
hypertensive cafeteria-fed rat model in which the renal
2A-adrenoceptor subtype is no longer
pharmacologically detectable in spite of unaltered gene expression when
compared with normal chow-fed rats (Coatmellec-Taglioni et al., 2000).
Indubitably, the discrepancy between gene expression and undetectable
2A-adrenoceptor sites in hypertensive rats
remains to be clarified. However, from these studies, it seems clear
that high-renal 2B-adrenoceptor densities do
not appear to be solely implicated in the hypertensive phenotype. It is
indeed possible that high-2B-adrenoceptor
densities associated with a lack of adequately functional renal
2A-adrenoceptor may facilitate the onset of
hypertension. Our present observations in SBN rats provide strong
support to this hypothesis. As a matter of fact, a high-salt diet
increases 2B-adrenoceptor densities in the
kidney of SBN rats but fails to induce any change in blood pressure. As
a contrast to SBH rats, high-salt diet markedly increases renal
2A-adrenoceptor mRNA and densities in SBN. It
is thus tempting to speculate about the potential significance of this
interstrain difference for renal
2-adrenoceptors in terms of genetic
predisposition to salt sensitivity and development of hypertension in
Sabra rats.
In normotensive rats, it has been shown that stimulation of the renal
2A-adrenoceptor subtype by the selective
2A-agonist guanfacine increases urine flow
rate solely by increasing osmolar clearance (Intengan and Smyth,
1997a). In spontaneously hypertensive rats, however, guanfacine fails
to increase osmolar clearance (Intengan and Smyth, 1997b), suggesting a
defect in renal 2A-adrenoceptor capacity. When
compared with SBN rats, SBH rats have a lower fractional sodium
excretion in the presence of comparable free water clearance (Ben-Ishay
and Yagil, 1994). Based on the natriuretic activity of the renal
2A-adrenoceptor shown in Wistar and
Sprague-Dawley rats (Intengan and Smyth, 1996, 1997a), it can
reasonably be postulated that the lack of this receptor in the SBH rats
may be contributing to the sensitivity to sodium and thus predisposing
these animals to develop hypertension when submitted to high-salt
intake. In contrast, 2A-adrenoceptors present
in the kidney of SBN rats are markedly increased by high-salt regimen.
This phenomenon could be an protective and adaptive change in these
animals in response to salt overload, resulting in an increase in
sodium excretion and the maintenance of normal blood pressure when
submitted to high-salt diet. However, this hypothesis must be supported
by investigating the function of renal
2A-adrenoceptors in Sabra rats. Evidently, the
key question that remains to be answered is whether these alterations
in renal 2-adrenoceptors are relevant to the
pathogenesis of hypertension in SBH rats. In fact, from a subsequent
series of studies on salt-induced hypertension in mice deficient in
each one of these 2-adrenoceptor subtypes
(Makaritsis et al., 1999a, 1999b, 2000) it has been suggested that
adrenergically mediated hypertension is a function of the
central 2B-adrenoceptor. Since we have shown
central differences in 2-adrenoceptor
densities between SBH and SBN rats (Parini et al., 1986), an
investigation of the distribution of their subtypes merits future studies.
In conclusion, these results demonstrate that marked differences exist
in expression levels of renal 2-adrenoceptor
subtypes between salt-sensitive and salt-resistant Sabra rats. If
high-renal 2B-adrenoceptor densities most
probably contribute to the hypertensive phenotype, results of the
present study also suggest that they are not implicated alone in the
salt-induced hypertension of the SBH rat. In fact, the absence of the
2A-subtype in SBH could also be responsible of
the lack of adequate receptor-mediated renal functions, predisposing
this rat substrain to the onset of hypertension under high-salt diet.
In contrast, the presence of the 2A-subtype in
SBN and its up-regulation under high-salt diet could protect this rat
strain against the pressive effect of high-salt diet and
consequently may explain the peculiar resistance to salt-induced
hypertension of these rats
SBH, salt-sensitive Sabra rats;
SBN, salt-resistant Sabra rats;
RT-PCR, reverse transcription-polymerase
chain reaction;
PCR, polymerase chain reaction;
bp, base pair(s).
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2A-Adrenoceptors Is
Associated with Resistance to Salt-Induced Hypertension in Sabra Rats
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Abstract
Introduction
