Upregulation of endothelin B receptors in kidneys of DOCA-salt
hypertensive rats
David M.
Pollock1,
Graham H.
Allcock1,
Arthi
Krishnan1,
Brian D.
Dayton2, and
Jennifer S.
Pollock1
1 Vascular Biology Center, Departments of
Surgery, Physiology and Endocrinology, and Pharmacology and
Toxicology, Medical College of Georgia, Augusta, Georgia
30912-2500; and 2 Pharmaceutical Discovery,
Abbott Laboratories, Abbott Park, Illinois 60064-3500
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ABSTRACT |
Experiments were designed to elucidate
the role of endothelin B receptors (ETB) on arterial
pressure and renal function in deoxycorticosterone acetate (DOCA)-salt
hypertensive rats. Male Sprague-Dawley rats underwent uninephrectomy
and were treated with either DOCA and salt (0.9% NaCl to drink) or
placebo. DOCA-salt rats given the ETB-selective antagonist,
A-192621, for 1 wk (10 mg · kg
1 · day
1
in the food) had significantly greater systolic arterial pressure compared with untreated DOCA-salt rats (208 ± 7 vs. 182 ± 4 mmHg) whereas pressure in placebo rats was unchanged. In DOCA-salt, but not
placebo rats, A-192621 significantly decreased sodium and water
excretion along with parallel decreases in food and water intake. To
determine whether the response in DOCA-salt rats was due to increased
expression of ETB receptors, endothelin receptor binding
was performed by using membranes from renal medulla. Maximum binding
(Bmax) of [125I]ET-1,
[125I]ET-3, and
[125I]IRL-1620 increased from 227 ± 42, 146 ± 28, and 21 ± 1 fmol/mg protein, respectively, in
placebo rats to 335 ± 27, 300 ± 38, and 61 ± 6 fmol/mg protein,
respectively, in DOCA-salt hypertensive rats. The fraction of receptors
that are the ETB subtype was significantly increased in
DOCA-salt (0.88 ± 0.07) compared with placebo (0.64 ± 0.01). The
difference between [125I]ET-3 and
[125I]IRL-1620 binding is consistent with
possible ETB receptor subtypes in the kidney. These results
indicate that ETB receptors in the renal medulla are
up-regulated in the DOCA-salt hypertensive rat and may serve to
maintain a lower arterial pressure by promoting salt and water excretion.
endothelin B receptors; deoxycorticosterone; hypertension; sodium
diet
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INTRODUCTION |
IN GENERAL, ETA and ETB
receptors produce vasoconstriction and vasodilation,
respectively. Administration of exogenous endothelin-1 (ET-1) activates
both receptor subtypes although ETA receptor-mediated vasoconstriction predominates (7, 36). The function of ETB receptors appear more complex as they are located on a variety of cell
types (29). On vascular endothelium, ETB receptors
stimulate release of nitric oxide to produce a transient vasodilation
(7, 11). ETB receptors are in particular abundance in renal
medullary epithelium, where they appear to influence tubular sodium and water reabsorption accounting for the natriuretic and diuretic actions
of ET-1 (9, 13, 20, 38). In addition, the ETB receptors are
thought to remove ET-1 from the circulation since blockade of
ETB receptors increases circulating levels of ET-1 (12,
24).
Plasma and tissue levels of ET-1 as well as mRNA expression have been
reported to be elevated in rats treated with DOCA (deoxycorticosterone acetate) and salt (1, 21, 22). Several laboratories including our own
have demonstrated that ET-1 contributes to the hypertension associated
with this model because selective ETA and non-selective ETA/ETB receptor antagonists lower arterial
pressure (1, 2, 23). However, we have recently observed that
ETA receptors have no sustained role in regulating renal
function (1). It is not clear what role the renal ETB
receptors may play under these circumstances of salt loading and high
ET-1 levels. We hypothesize that ETB receptors located
within renal medulla may serve to promote salt and water excretion
under conditions of a high salt load.
The purpose of the present study was to 1) determine the
functional role of ETB receptors in DOCA-salt hypertensive
rats and 2) determine the influence of DOCA-salt treatment on
ETB receptor expression in the renal medulla. A recently
available ETB-selective receptor antagonist, A-192621
(34a), was used to determine the effect of ETB receptor
blockade on arterial pressure and renal sodium and water handling in
DOCA-salt, hypertensive rats. ETB receptor mRNA and protein
expression were determined by Northern blot and receptor-binding assay,
respectively. We describe a new method for performing
radioligand-binding studies requiring no separation of bound and free
ligand that can be used for quantitating receptor number and kinetic
analysis in tissues(17, 34).
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METHODS |
Experiments were conducted by using male Sprague-Dawley rats with an
initial body weight of 200-220 g (Harlan Laboratories, Indianapolis, IN). Animal protocols were in accordance with National Institutes of Health guidelines and approved and monitored by the
Medical College of Georgia Committee for Animal Use in Research and
Education. Rats were housed under conditions of constant temperature and humidity and exposed to a 12:12-h light-dark cycle. After adjusting
to these conditions for several days, rats were anesthetized with
methohexital sodium (50 mg/kg, ip, Brevital, Eli Lilly, Indianapolis, IN), and a right nephrectomy was performed via a retroperitoneal incision. A 60-day time-release pellet of DOCA (200 mg) or placebo (Innovative Research of America, Sarasota, FL) was also implanted subcutaneously. After recovery, rats receiving the DOCA pellet were
given 0.9% NaCl to drink whereas placebo rats were given tap water.
ETB blockade in DOCA-salt and placebo rats.
DOCA-salt and placebo rats were maintained in metabolic cages to allow
for urine collection and measurement of food and water intake. One week
after the surgery as described above, hypertension was verified in the
DOCA-salt rats by measuring arterial pressure by the tail cuff method
(1). Separate groups of DOCA-salt and placebo-treated rats were then
given a selective ETB receptor antagonist, A-192621 (Abbott
Laboratories, Abbott Park, IL), in the food (n = 5 and 6, respectively) whereas additional DOCA-salt and placebo rats were
continued on normal chow (n = 5 and 6, respectively). The
concentration of A-192621 was adjusted daily to maintain a dose of 10 mg · /kg
1 · day
1.
This dose and delivery scheme has been shown to produce complete inhibition of ETB-induced hypotension produced by
sarafotoxin 6c in normal rats (34a). After 1 wk of treatment with the
antagonist, urine was collected and food and water intake were measured
over a 24-h period, followed immediately by another arterial pressure measurement. Rats were then anesthetized with pentobarbital sodium, and
a terminal blood sample was taken from the abdominal aorta for plasma
creatinine determination. Electrolyte content in urine was measured by
the Synchron EL-ISE electrolyte system (Beckman Instruments, Brea, CA).
Urinary and plasma creatinine was measured by the picric acid method
adapted for microtiter plates (1).
ETB receptor mRNA expression.
Three weeks after DOCA and placebo pellets were implanted, rats were
anesthetized with pentobarbital sodium (65 mg/kg, ip, Abbott
Laboratories, North Chicago, IL), kidneys were excised, dissected into
cortex and medulla, and frozen under liquid nitrogen and stored at
80°C. Tissue was pulverized while still frozen, and total
RNA was extracted by a guanidine isothiocyanate-phenol-chloroform method using TRIzol reagent (4). Total RNA samples (20 µg) were made
up to equal volumes in nuclease-free diethylpyrocarbonate (DEPC) water
and then incubated at 65°C for 5 min with 10× MOPS, 37%
formaldehyde, and formamide. Ten microliters of 5× glycerol loading dye and 1 µl of ethidium bromide (10 mg/ml) were
then added, and the samples were mixed before loading onto a 1%
agarose gel containing 72 ml DEPC water, 10 ml 10× MOPS, and 18 ml formaldehyde. The samples were run on the gel surrounded by 1×
MOPS at 120 V for 1.5-2 h. The samples were then transferred from
the gel to a positively charged nylon membrane (TotalBlot+, Amresco,
Solon, OH) by capillary action with 3 mol/l NaCl, 0.3 mol/l sodium
citrate (20× SSC) overnight. Membranes were washed with 2×
SSC for 10 min and then allowed to dry before locations of the 18S and
28S rRNA species were revealed by observing the ethidium bromide
staining under ultraviolet light. Membranes were prehybridized at
42°C for 3 h in 2× SDS, 50% formamide, and a
prehybridization solution containing 12× SSC, 10×
Denhardt's solution, and 200 µg/ml sheared, denatured salmon sperm.
The membranes were hybridized for 18-20 h at 42°C in the above
prehybridization solution with 10% dextran sulfate and 25 ng of the
32P-labeled probe. The probe p3B-3 is a 511-base-pair
fragment of rat ETB receptor that had been cut out of a
cloned vector pGEM-3z (Promega) by using BamHI. The membranes
were then washed twice for 30 min in 2× SSC, 1% SDS at 55°C,
and finally for 30 min in 0.2× SSC, 0.1% SDS at 55°C.
Membranes were dried and then exposed to autoradiographic film
(DuPont-NEN, Boston, MA) for 1-4 days. Autoradiograms and the
ethidium bromide staining of the 18S and 28S rRNA bands were analyzed
by densitometry (IS-1000 digital-imaging system, Alpha Innotech, San
Leandro, CA).
Receptor-binding assay.
Binding characteristics of [125I]ET-1,
[125I]ET-3, and
[125I]IRL-1620 were determined by using
membrane preparations obtained from the rat renal medulla.
[125I]ET-1 binding represents the total number
of ETA and ETB receptors whereas
[125I]ET-3 and
[125I]IRL-1620 bind exclusively to
ETB receptors. Three weeks after DOCA and placebo pellets
were implanted, rats were anesthetized and kidneys were excised,
dissected, and frozen as described above for mRNA isolation. To prepare
membranes for the binding assay, tissue was first weighed and
pulverized. The pulverized tissue was then added to homogenization
buffer [250 mM sucrose, 50 mM Tris · HCl, pH
7.4, 5 mM EDTA, and 15 µM phenylmethylsulfonyl (PMSF)] in a
glass/Teflon homogenizer at a ratio of ~1 g tissue/5-10 ml
buffer. The tissue was then homogenized for 20 strokes. The homogenate
was centrifuged at 1,000 g for 30 min at 4°C. The resulting supernatant was centrifuged at 30,000 g for 45 min at 4°C.
This supernatant was removed, and the pellet was resuspended in
one-half the initial amount of homogenization buffer. The protein
concentration was then assessed by the Bradford method (Bio-Rad,
Hercules, CA).
A known quantity of each membrane preparation was added to each well of
a 96-well microtiter plate (Optiplate, Packard Instruments, Meridan,
CT). Wheat germ agglutinin polyvinyltoluene beads (scintillation proximity beads, Amersham Life Science, Arlington Heights, IL) were
suspended in binding buffer (40 mg/ml) and 1 mg was added to each well.
Binding buffer was composed of (in mM) 20 Tris, 100 NaCl, and 10 MgCl2, pH 7.4, and contained 0.1 mM PMSF, 5 µg/ml pepstatin A, 0.025% bacitracin, 3 mM EDTA, and 0.2% BSA. The plate was covered and shaken gently for 2.5 h at room temperature. After this
precoupling process, 25 µl of binding buffer were added to those
wells required for total binding, whereas ET-1 was added to the other
wells (final concentration of 1 µM) for the nonspecific binding.
[125I]ET-1 was diluted in binding buffer and
then added to each well for each ligand concentration on the binding
curve. The plate was sealed and shaken gently for 18 h at room
temperature. The plate was then centrifuged at 1,000 g for 5 min before being counted on a Packard TopCount scintillation counter.
Before a binding curve was established, the amount of protein required
was estimated by performing a protein curve (2-100 µg) using
membranes obtained from renal medulla of normal, placebo, and DOCA-salt
rats. Also, the optimum time needed for equilibration was also
established by examining a range of times (2-24 h). Total and
nonspecific binding were assessed for 1 nM
[125I]ET-1 at each of the protein
concentrations. To assess ETB receptor binding, the
radioligands [125I]ET-3 and
[125I]IRL-1620 were utilized in the same
protocol. ET-1 was used (final concentration of 1 µM) to assess
nonspecific binding with the ligand [125I]ET-3,
whereas IRL-1620 was utilized (final concentration of 1 µM) with the
ligand [125I]IRL-1620 to assess nonspecific
binding. All points were performed in duplicate, and all dilutions of
peptides were performed in siliconized tubes.
Materials.
Wheat germ agglutinin scintillation proximity assay (SPA) beads were
obtained from Amersham Life Sciences.
[125I]ET-1 (2,200 Ci/mmol),
[125I]ET-3 (2,200 Ci/mmol), and
[125I]IRL-1620 (2,200 Ci/mmol) were purchased
from New England Nuclear. ET-1 and IRL-1620 were obtained fron American
Peptide (Sunnyvale, CA). MOPS (10×) , 20× SSC, formamide,
formaldehyde, TotalBlot+ nylon membranes, and agarose were all obtained
as part of the TotalBlot Northern kit (Amersco). SDS, 5× glycerol
gel loading buffer, and DEPC-treated nuclease-free water were also
obtained from Amersco. TRIzol reagent and 2×
prehybridization/hybridization solution came from GIBCO-BRL (Grand
Island, NY). [
-32P]-dCTP was obtained from
Amersham. PMSF, pepstatin A, and bacitracin were purchased from Sigma
Chemical (St. Louis, MO). All other agents were obtained from Bio-Rad Laboratories.
Statistical analysis.
Analysis of variance with Fisher's protected least-significant
differences post hoc test was used for statistical evaluation of data.
Values are reported as means ± SE, with P < 0.05 being considered significant. Binding data were analysed by nonlinear regression of the binding isotherm (Prism, GraphPad Software, San
Diego, CA). Scatchard analysis is also shown for historical comparison
purposes only.
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RESULTS |
One week after DOCA or placebo pellets were implanted, systolic
arterial pressure was significantly greater in DOCA-salt rats (156 ± 4 mmHg) compared with placebo-treated rats (141 ± 2 mmHg, P < 0.05). The effect of ETB receptor blockade on systolic
arterial pressure and creatinine clearance in placebo and DOCA-salt
treated rats is presented to Fig. 1.
A-192621 treatment for 1 wk significantly increased arterial pressure
in DOCA-salt but not placebo rats (Fig. 1A). Creatinine
clearance was reduced in DOCA-salt rats compared with placebo and was
unaffected by ETB receptor blockade (Fig. 1B). As
expected, sodium excretion and urine volume were significantly
increased in DOCA-salt rats compared with placebo (Fig.
2). Treatment with A-192621 produced a
signficant reduction in sodium excretion and urine volume in DOCA-salt
but not placebo rats. Neither food nor water intake was affected by
ETB receptor blockade in placebo rats (Fig.
3). Food intake was slightly but significantly lower in DOCA-salt rats compared with placebo (Fig. 3A). Water intake was significantly increased in DOCA-salt rats compared with placebo (Fig. 3B). Sodium and water balance,
calculated as the difference between intake and urinary excretion, was
significantly greater in DOCA-salt rats compared with placebo (Table
1). ETB blockade significantly
reduced sodium and water balance in DOCA-salt but not placebo rats; the
decrease in water balance in DOCA-salt rats was to levels even lower
than placebo rats. It should be noted that the mode of calculation of
water balance did not take into account extrarenal losses of water.
This probably explains why we found a positive water balance in placebo
rats instead of zero.

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Fig. 1.
Effect of endothelin B (ETB) receptor antagonist A-192621
on systolic pressure (A) and creatinine clearance (B)
in placebo and deoxycorticosterone acetate (DOCA)-salt rats (n = 6 in both placebo groups and n = 5 in both DOCA-salt groups).
Values are means ± SE. * P < 0.05 compared with placebo
rats. P < 0.05 compared with untreated DOCA-salt
rats.
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Fig. 2.
Effect of ETB receptor antagonist A-192621 on sodium
excretion (A) and urine volume (B) in placebo and
DOCA-salt rats. Values are means ± SE. * P < 0.05 compared with placebo rats. P < 0.05 compared with
untreated DOCA-salt rats.
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Fig. 3.
Effect of ETB receptor antagonist A-192621 on food intake
(A) and water intake (B) in placebo and DOCA-salt rats.
Values are means ± SE. * P < 0.05 compared with placebo
rats. P < 0.05 compared with untreated DOCA-salt rats.
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Table 1.
Effect of 1-wk treatment with ETB receptor antagonist
A-192621 on sodium and water balance in DOCA-salt and placebo rats
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Northern blot analysis was utilized to determine whether the increased
response to ETB receptor blockade in vivo could be accounted for by changes in mRNA expression. ETB receptor
mRNA levels were approximately two times greater in tissue from medulla compared with cortex (Fig. 4). However,
DOCA-salt treatment did not affect these ETB mRNA levels
compared with placebo controls.

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Fig. 4.
A: representative Northern blot for ETB receptor
mRNA isolated from cortical and medullary renal tissue of DOCA-salt and
placebo rats. P, placebo rats; D, DOCA-salt rats. B: blots were
scanned, and relative intensities to 18S ribosomal RNA band were
obtained from 5 rats in each group. Data represent means ± SE.
ETB mRNA levels were significantly greater in medulla
compared with cortex for both groups (P < 0.05).
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Receptor-binding studies were conducted to determine whether increased
ETB receptor protein could account for the responses to
ETB receptor blockade in DOCA-salt rats. The membrane
containing receptor protein was isolated from renal medullary tissue
and bound onto the wheat germ agglutinin beads, which then scintillates only when radioactive ligand is bound. This newly developed
scintillation proximity assay (SPA) has the technical advantage of not
having to separate bound ligand from free (17, 34). Initial binding experiments were conducted to characterize the method by using renal
medullary tissue obtained from normal Sprague-Dawley rats. Total and
nonspecific bindings were determined for
[125I]ET-1 over a range of membrane protein
concentrations (Fig. 5). Maximum binding
was achieved at 20 µg/well; therefore, this concentration was used in
subsequent experiments. Optimal equilibration time for all three
ligands was found to be 18 h. Saturation binding curves and Scatchard
plots were then obtained for each ligand, [125I]ET-1, [125I]ET-3,
and [125I]IRL-1620, by using membrane
preparations from normal rats (Figs. 6).
Similar to what has been reported using traditional methods of receptor
binding (27), the SPA technique indicated that the renal medulla
contains ~32% ETA and 68% ETB receptors.
Maximum binding of the ETB ligand,
[125I]ET-3, was greater than that of the
ETB ligand, [125I]IRL-1620.

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Fig. 5.
Total and nonspecific binding of [125I]ET-1 in
membrane preparations from renal medulla of normal Sprague-Dawley rats
(n = 3). Values are means ± SE. CPM, counts/min.
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Fig. 6.
Saturation binding isotherms and Scatchard analysis (insets) of
[125I]ET-1 (A),
[125I]ET-3 (B), and
[125I]IRL-1620 (C) in membrane
preparations from renal medulla of normal Sprague-Dawley rats
(n = 5, 3, and 3, respectively).
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Maximum binding (Bmax = fmol/mg protein) of
[125I]ET-1, [125I]ET-3,
and [125I]IRL-1620 in membrane preparations
from renal medulla of placebo and DOCA-salt hypertensive rats are
presented in Fig. 7 (n = 6 for all
3 ligands). Total and nonspecific bindings were determined for
[125I]ET-1 over a range of membrane protein
concentrations from both placebo and DOCA-salt rat kidneys. Maximum
binding was achieved at 20 µg of protein/well for placebo kidneys
similar to kidneys from normal Sprague-Dawley rats, whereas 10 µg of
protein/well achieved maximum binding for DOCA-salt kidneys. Bindings
of [125I]ET-1,
[125I]ET-3, and
[125I]IRL-1620 were increased by ~45, 105, and 180%, respectively, in the DOCA-salt hypertensive rat compared
with placebo. The increase observed for
[125I]ET-1 was of borderline significance
(P = 0.051). The ratios of the maximum number of binding sites
for ET-3 to ET-1, IRL-1620 to ET-1 and IRL-1620 to ET-3 are presented
in Fig. 8. The increase in ET-3/ET-1 ratio
indicates that ETB receptors were increased whereas
ETA receptors appear to be decreased in the DOCA-salt hypertensive rat. For ETB receptors, IRL-1620 binding
represents only ~20% of the total number of ETB
receptors. DOCA-salt treatment had no effect on IRL-1620 binding as a
proportion of the total number of ETB receptors. In other
words, both IRL-1620-sensitive and -insensitive ETB
receptor binding was increased in DOCA-salt rats. Dissociation
constants (KD; nM) for
[125I]ET-1, [125I]ET-3,
and [125I]IRL-1620 binding in medullary
membrane preparations were not significantly different between the
placebo and DOCA-salt rat (Table 2).

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Fig. 7.
Maximum binding (Bmax) of
[125I]ET-1, [125I]ET-3,
and [125I]IRL-1620 in membrane preparations
from renal medulla of placebo and DOCA-salt rats (n = 6/group). Values are means ± SE. * P = 0.051 compared with
placebo rats. P < 0.05 compared with placebo rats.
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Fig. 8.
Ratio of total binding of ET-3 to ET-1, IRL-1620 to ET-1, and IRL-1620
to ET-3 in membrane preparations from renal medulla of placebo and
DOCA-salt rats * P < 0.05 compared with placebo control.
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Table 2.
Dissociation constants (KD, nM) of
[125I]ET-1, [125I]ET-3 and
[125I]IRL 1620 binding in membrane preparations from
renal medulla of placebo and DOCA-salt rats
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DISCUSSION |
The recent availability of novel, orally active ETB
receptor-selective antagonists has greatly increased the interest and ability to investigate the physiological role of the ETB
receptor. In our experiments, the ETB receptor antagonist
A-192621 was administered chronically to DOCA-salt and placebo rats.
One week of treatment with A-192621 increased arterial pressure in
DOCA-salt hypertensive rats but had no effect on placebo rats. These
findings suggest that ETB receptors play an important role
in counteracting hypertension in the DOCA-salt rat. Acute
administration of the ETB antagonists Ro-46-8443 or BQ-788
increased arterial pressure in DOCA-salt rats, similar to our findings
during chronic ETB receptor blockade (5, 18). In contrast,
Ro-46-8443 actually decreased arterial pressure in normotensive
Wistar-Kyoto rats but only at a higher dose (5). With chronic
administration, we observed that ETB blockade had no effect
on arterial pressure in normotensive rats. The reason for this apparent
discrepancy in normal rats may be due to any number of factors
including the dose of antagonist and the duration of treatment. The
role of the ETA receptor appears to be quite different from
that of the ETB receptor. Chronic ETA receptor
blockade has no effect on arterial pressure in normal rats but clearly
attenuates the hypertension in DOCA-salt rats (1, 32). Taken together
with findings that ET-1 production is elevated in the DOCA-salt rat, a
complex role for ET-1 has emerged in the regulation of vascular and
renal function. The functional consequences may be to promote
natriuresis and diuresis by elevating renal perfusion pressure via
ETA receptors while the ETB receptor inhibits
sodium and water reabsorption directly within the kidney.
Matsumura et al. (25) recently reported that renal vasoconstriction and
aortic vascular hypertrophy were exaggerated in DOCA-salt hypertensive
rats as a result of a 2-wk treatment with A-192621 at 30 mg · kg
1 · day
1.
In contrast to our findings, these investigators did not observe any
influence of ETB receptor blockade on arterial pressure.
The reason for the different findings is not clear. We used a slightly lower dose of A-192621 administered in the food compared with twice
daily oral gavage in the study of Matsumura et al. Although both
studies initiated treatment before the DOCA-salt rats reached maximum
hypertension, arterial pressure was higher both before and after
treatment with the antagonist in the present study. It is possible that
differences in arterial pressure before dosing and/or differences in
rat strains could explain the disparate results.
We hypothesized that the increase in sensitivity to ETB
receptor blockade in DOCA-salt rats may be due to increased
ETB receptor gene expression. Because the kidney contains
the highest concentration of ETB receptors of any tissue
(29), we determined ETB mRNA expression in the renal cortex
and medulla of DOCA-salt and placebo rats. The medulla contained larger
amounts of ETB mRNA compared with cortex, similar to what
has been reported for ETB receptor protein by using
autoradiographic and competitive binding methods (8, 19, 27). However,
we were unable to discern a difference in ETB mRNA between
the hypertensive and normotensive rats. These results would suggest
that DOCA-salt hypertension does not increase gene expression, which
then leaves open the question as to how ETB receptors serve
to lower pressure in these rats.
Although ETB mRNA may not be changed in DOCA-salt
hypertension, it is still possible that posttranscriptional events may
allow for an increase in the actual number of ETB receptors
available for binding ET-1 in the DOCA-salt rat. Using a newly
developed binding assay, we were able to confirm previous reports using kidneys from normotensive rats that ~70% of the total number of ET-1-binding sites in the renal medulla are of the ETB
variety. In the renal medulla of DOCA-salt rats, ETB
receptors were increased by roughly twofold compared with placebo.
IRL-1620 and ET-3 binding were increased to a similar extent.
ETA receptors appeared to be reduced because the total
number of binding sites did not increase to the same extent as did the
ETB-binding sites. The apparent decrease in
ETA-binding sites is consistent with the observation that
ETA-induced vasoconstriction is significantly attenuated in
isolated small arteries of the DOCA-salt rat (14). Although we observed
significant increases in ETB receptor expression, this may
not be unique to the DOCA-salt rat. Further studies are necessary to
determine whether increases in sodium intake alone would alter
ETB receptor expression.
The contrasting findings suggest that mRNA expression for
ETB receptors was unchanged whereas the total number of
binding sites was increased suggests that ETB receptor
protein expression is most likely regulated at the posttranscriptional
level. Alternatively, it is possible that mRNA stability is reduced in
DOCA-salt kidneys or that Northern blot analysis is not sensitive
enough to detect these changes. The latter possiblity seems unlikely
becasue we were able to discern a twofold difference in ETB
mRNA expression between medulla and cortex.
Although both ET-3 and IRL-1620 are selective for the ETB
receptor in this assay, the number of binding sites was considerably less for IRL-1620. Nambi, Brooks, and colleagues (3, 26) have
previously observed IRL-1620-sensitive and -insensitive ETB receptors and have suggested that these may represent ETB1
and ETB2 receptor subtypes. By definition, the
ETB1 receptor subtype is found on endothelial cells and
produces vasodilation via nitric oxide release (15, 30, 31, 35). The
ETB2 receptor, on the other hand, is found on vascular
smooth muscle and produces vasoconstriction. IRL-1620 reportedly binds
to ETB receptors found on both endothelial cells and
vascular smooth muscle (33) and can produce medullary vasodilation and
cortical vasoconstriction within the rat kidney (10, 37). Therefore,
the evidence argues against the possibility that the differences in
IRL-1620 and ET-3 distinguish ETB1 and ETB2
binding. An alternate, yet untested, hypothesis is that these ligands
could distinguish between ETB receptors found on the
vascular endothelium vs. those on the tubular epithelium because
ETB receptors are located on both cell types within the
renal medulla. In any event, it is clear that both IRL-1620-sensitive
and -insensitive bindings are elevated in this model.
There are several possible mechanisms by which ETB
receptors can influence arterial pressure. First, ETB
receptors have been proposed to function as regulators of ET-1 activity
by binding and removing ET-1 from the circulation (7, 12). It is
possible that the increased ETB receptor expression is in
response to elevated production of ET-1 as previously reported in
DOCA-salt rats (1, 6, 21, 22). However, ET-1 is primarily cleared by
ETB receptors in the lungs (12), so it would appear
unlikely that the increase in renal ETB receptors plays a
similar role. There is also good evidence that ETB
receptors located on the vascular endothelium can stimulate nitric
oxide release and function to oppose the vasoconstrictor actions of
ET-1 mediated through the ETA receptor. Infusion of ET-1
produces only a transient vasodilation, which is then followed by a
prolonged vasoconstriction. This has led many to speculate that
ETB-mediated vasodilation plays only a minor role in
mediating ET-1-dependent vascular tone (7, 36). Although ET-1 infusion
does not produce any vasodilation in the rat in terms of whole kidney
blood flow, it does produce increases in medullary blood flow
simultaneous with decreases in cortical blood flow (16). Therefore,
ETB receptors may stimulate vasodilation of the medullary
circulation as a "washout" phenomenon, similar to what has been
proposed for many vasodilators. Finally, there is also abundant
evidence that ETB receptors located on collecting duct
cells can inhibit tubular reabsorption directly (9, 13, 20, 38), which
in turn could decrease arterial pressure through changes in
extracellular fluid volume.
The DOCA-salt rat is characterized by an increase in sodium and water
balance, i.e., sodium and water retention. ETB receptor blockade significantly reduced sodium and water balance in DOCA-salt rats. Certainly, the accompanying reduction in sodium and water intake
may account for this effect. It is not clear how water intake would be
decreased directly by ETB receptor blockade, but an effect
to inhibit thirst is not unreasonable to propose because water balance
was actually below that of placebo in DOCA-salt rats treated with
A-192621. It is possible that the influence of ETB
receptors on thirst mechanisms may be evident only during a severe
stimulus for increasing water intake, such as DOCA-salt treatment. We
expected that ETB receptor blockade would have increased reabsorption of sodium and water because ETB receptor
activation has been shown to inhibit sodium and water reabsorption in
vitro (13, 20). On the other hand, our observations indicate an inappropriately low level of sodium excretion given the increase in
renal perfusion pressure. Similar observations have been made during
acute ETB receptor blockade where pressure was increased yet excretion remained unchanged (18). Thus ETB receptors
may be important in mediating pressure natriuresis. Nonetheless, more definitive studies on the role of ETB receptors in the
physiological regulation of sodium and water balance are clearly warranted.
In conclusion, these results support the hypothesis that
ETB receptors serve to attenuate hypertension in DOCA-salt
rats. The additional sodium and water load that exists in the DOCA-salt rat appears to reveal a critical role for the ETB receptor.
Although there are several mechanisms by which this can be
accomplished, we have provided evidence that the number of
ETB receptors is elevated in the renal medulla in this
model. Although ETB receptors may serve to increase removal
of ET-1 from the circulation and stimulate endothelial-dependent
vasodilation, the upregulation of renal medullary ETB
receptors is consistent with their proposed role in promoting sodium
and water excretion.
 |
ACKNOWLEDGEMENTS |
The authors thank Jyoti Thakkar and Deborah Garner for expert
technical assistance.
 |
FOOTNOTES |
These studies were supported by the generosity of Abbott Laboratories
for supplying the scintillation proximity assay beads and A-192621 (J. L. Wessale and T. J. Opgenorth) and by American Heart Association
Scientist Development Grants (D.M. Pollock and J. S. Pollock).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. M. Pollock,
Vascular Biology Center, Medical College of Georgia, Augusta, GA
30912-2500 (E-mail: dpollock{at}mail.mcg.edu).
Received 5 March 1999; accepted in final form 14 September 1999.
 |
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