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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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). [alpha -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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. dagger  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. dagger  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. dagger 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

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).

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).

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. dagger  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


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Physiol 278(2):F279-F286
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