INTRODUCTION

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IN 1988, YANAGISAWA ET AL. (33) isolated a potent vasoconstrictor produced by endothelial cells that was named endothelin (ET). Inoue and colleagues (14) determined through DNA analysis that there are three distinct genes encoding three ET isopeptides (ET-1, ET-2, and ET-3). G protein-coupled receptors were discovered and then characterized by their selective affinity to each of the ET isopeptides, location, and their biological function (18). Endothelin A (ET_A) receptors bind ET-1 with higher affinity than ET-2 or ET-3, are localized to vascular smooth muscle cells, and cause vasoconstriction. Endothelin B (ET_B) receptors are found on endothelial cells and have been shown to produce vasodilation and bind all three ET isopeptides with equal affinity. Additionally, there is evidence that ET_B receptors in pulmonary endothelial cells "clear" ET-1 from the circulation (7). However, this classification of ET receptors may be oversimplified, with increasing evidence suggesting additional receptor subtypes. It is now known that there exist two pharmacologically distinct subtypes of the ET_B receptor, ET_B1 and ET_B2, whose biological activity is a function of their localization. ET_B1 are located on the vascular endothelium and produce vasodilation upon agonist binding, whereas ET_B2 are found on vascular smooth muscle cells and mediate non-ET_A vasoconstriction.

Within the kidney, both ET receptor subtypes have been identified in various regions differing both in number and function. In both cortex and medulla, ET_B receptors are more abundant than ET_A receptors (24). The ET_B receptor has been localized to renal arterioles, glomeruli, proximal convoluted tubule, medullary thick ascending loop, and collecting duct (29). ET_B receptors in the renal vasculature couple to nitric oxide and prostaglandin synthesis, leading to vasorelaxation (6), whereas ET_B receptors on renal tubular cells promote natriuresis and diuresis (8, 20). ET_A receptors localized in arterioles and proximal tubules mediate vasoconstrictor (5, 20) and antinatriuretic effects (18, 26).

Elevated plasma levels of ET-1 have been implicated in the pathogenesis of many disease models, including pulmonary hypertension (2) and DOCA salt hypertension (27). Although plasma ET-1 levels are high in these disease models, there is still conflicting evidence as to the regulation of ET receptors. Studies by Bauer et al. (2) have shown increased mRNA transcripts encoding the ET_B receptor with no difference in ET_A mRNA levels in pulmonary arterial tissue taken from patients with pulmonary hypertension. However, in endothelium-denuded saphenous veins from African-American patients with essential hypertension, elevated plasma ET-1 levels may be responsible for a decrease in ET_A mRNA expression together with increased ET_B mRNA levels (13). In DOCA-salt hypertensive rats, Pollock et al. (27) observed increased ET_B receptor number with an apparent decrease in ET_A receptor number in the renal medulla. Conversely, a transient elevation of plasma ET-1 levels induced by adenovirus gene transfer led to systemic hypertension but no change in renal ET receptor number (28).

The spotting-lethal rat carries a natural 301-bp deletion in the ET_B receptor that abrogates expression of functional ET_B receptors. Rats that are homozygous (sl/sl) for this mutation exhibit a lethal phenotype of congenital intestinal agangliosis and are commonly used as a model for Hirschsprung's disease (9). In 1998, Gariepy et al. (11) used a dopamine--hydroxylase promoter to direct transgenic ET_B receptor expression in adrenergic tissue that "rescued" the rats from the intestinal defect. The sl/sl animals have elevated plasma ET-1 levels. Therefore, the purpose of this study was to determine whether ET_B receptor deficiency and elevated plasma ET-1 levels are associated with changes in ET receptor density in the kidney.

	METHODS

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Membrane preparations. All of the procedures carried out on the rats were approved by the Medical College of Georgia Animal Care and Use Committee and followed the guidelines of the American Physiological Society. Rats (300-350 g) heterozygous and homozygous for the ET_B receptor deficiency from our local breeding colony and male Sprague-Dawley (Harlan) rats (300-350 g) were anesthetized, and kidneys were excised and separated with a razor blade on ice into cortex, outer medulla, and inner medulla and frozen at 80°C. Tissue was weighed, pulverized, and then homogenized in buffer containing 250 mM sucrose, 50 mM Tris · HCl, 5 mM EDTA, and 15 µM phenylmethylsulfonyl fluoride (PMSF), pH 7.4, as described (27). Briefly, homogenized samples were then centrifuged at 1,000 g at 4°C for 30 min. The supernatant was further centrifuged at 30,000 g at 4°C for 45 min, and the resulting pellet was resuspended in one-half of the starting volume of homogenization buffer and frozen at 80°C. The protein concentration of the membrane preparations was determined by the Bradford method (Bio-Rad, Hercules, CA).

Saturation binding curves. Receptor binding curves were performed to quantify the amount of ET receptors in the cortex, outer medulla, and inner medulla of sl/+ or sl/sl rats for the ET_B receptor deficiency. Sprague-Dawley rat renal tissue was also used in preliminary experiments to serve as a comparison to both previously published data and the transgenic animals. Membrane preparations from each of the three renal tissue sections were bound to wheat germ agglutinin scintillation proximity beads as previously described (27). Briefly, membrane preparations of sl/+ or sl/sl ET_B receptor-deficient rats from renal cortex (20 µg), outer medulla (20 µg), or inner medulla (5 µg) were added to each well of a 96-well microtiter plate (Optiplate; Packard Instruments, Meridian, CT). Wheat germ agglutinin polyvinyltoluene beads (scintillation proximity beads; Amersham Life Science, Arlington Heights, IL) were suspended in binding buffer (40 mg/ml), with 1 mg added to each well. Binding buffer consisted of 20 mM Tris · HCl, 100 mM NaCl, 10 mM MgCl₂, and 3 mM EDTA, pH 7.4, with additional 0.1 mM PMSF, 5 µg/ml pepstatin A, 0.025% bacitracin, and 0.2% BSA. The plate was covered and shaken for 3 h at room temperature to allow for coupling of the protein to the beads. After this precoupling, 25 µl of binding buffer were added to wells for total binding determination, whereas 2 µM ET-1 or ET-3 was added to determine nonspecific binding. [¹²⁵I]ET-1 or [¹²⁵I]ET-3 was diluted in binding buffer and added to each well with a final concentration of 0.03, 0.1, 0.3, 1, or 3 nM. The plate was sealed and shaken for 18 h at room temperature. The plate was then centrifuged for 5 min at 1,000 g before being counted on a Packard TopCount microplate scintillation counter. Our laboratory had previously determined optimum incubation times and procedures (27). All measurements were performed in duplicate, and all dilutions of radioactive and nonradioactive peptides were made in siliconized tubes.

Competition binding curves. Competition binding curves were performed using selective ET_A and ET_B receptor antagonists. Preliminary experiments were performed using inner medullary tissue preparations from Sprague-Dawley animals. Membrane preparations from the inner medulla (5 µg) of sl/+ or sl/sl animals were precoupled to scintillation proximity beads similar to the same receptor binding protocol described above. For generation of competition binding curves, either the ET_A receptor antagonist A-127722 (0.01 pM-0.5 mM), the ET_B receptor antagonist A-192621 (100 pM-10 mM), or unlabeled ET-1 (10 pM-1 µM) was added to separate wells followed by the addition of [¹²⁵I]ET-1 (1 nM) to every well. The plate was shaken for 18 h and then counted on the microplate scintillation counter.

Determination of maximal binding and dissociation constant values. Experiments using Chinese hamster ovary cells stably transfected with either the ET_A or ET_B receptor have shown that the ET_A receptor preferentially binds ET-1 over ET-3 (IC₅₀ values of 0.28 and 475 nM, respectively), whereas the ET_B receptor binds both ET-1 and ET-3 with similar affinity (IC₅₀ values of 0.14 and 0.08 nM, respectively; see Ref. 19). Thus, in our receptor binding experiments, [¹²⁵I]ET-3 receptor binding was used to determine maximal binding (B_max) values for ET_B receptor number. To determine ET_A receptor number, B_max values for [¹²⁵I]ET-3 binding were subtracted from [¹²⁵I]ET-1 B_max values.

Materials. Wheat germ agglutinin scintillation proximity assay beads were purchased from Amersham Life Sciences. [¹²⁵I]ET-1 (2,200 Ci/mmol) was obtained from New England Nuclear (Boston, MA), and [¹²⁵I]ET-3 (2,000 Ci/mmol) was acquired from Amersham Life Sciences. ET-1 and ET-3 were purchased from American Peptide (Sunnyvale, CA). A-127722 {trans-trans-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-([N,N-dibutylamino]carbonylmethyl)pyrrolidine-3-carboxylate} and A-192621 {[2R-(2,3,4)]-4-(1,3-benzodioxol-5-yl)-1-{2-[(2,6 diethylphenyl)amino}-2-oxoethyl]-2-(4-propoxyphenyl)-3-pyrrolidinecarboxylic acid} were obtained from Dr. Jerry Wessale at Abbott Laboratories (Abbott Park, IL). PMSF, pepstatin A, and bacitracin were purchased from Sigma Chemical (St. Louis, MO). All other agents were acquired from Bio-Rad Laboratories.

Data and statistical analysis. Receptor binding data were analyzed by nonlinear regression using the one-site model of the binding isotherm except for the Scatchard analysis of [¹²⁵I]ET-1 binding in inner medulla of sl/sl animals in which a two-site model was utilized (Prism; GraphPad Software, San Diego, CA). Statistical differences in the mean values for all binding data were determined by ANOVA, and Fisher's protected least-significant difference test was used to determine differences between individual means (StatView, Abacus Concepts, Berkley, CA). Values are reported as means ± SE, with P < 0.05 and P < 0.001 considered significant.

	RESULTS

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Saturation binding of [¹²⁵I]ET-1 in renal tissue. Total and nonspecific binding was determined for [¹²⁵I]ET-1 over a wide range of renal cortical, outer medullary, and inner medullary membrane protein concentrations. Maximum binding for sl/+ and sl/sl cortex and outer medulla preparations was achieved at 20 µg, whereas only 5 µg were needed for maximum binding in the inner medulla (data not shown). Maximum binding of [¹²⁵I]ET-3 in the inner medulla required only 0.5 µg for maximum binding (data not shown). Figure 1 shows saturation binding curves of cortex (20 µg), outer medulla (20 µg), or inner medulla (5 µg) of membrane preparations from sl/+ or sl/sl rats incubated with [¹²⁵I]ET-1 (1 nM). Specific binding of ET-1 was similar and saturable in both the renal cortex (Fig. 1A) and renal outer medulla (Fig. 1B) of sl/+ ET_B receptor-deficient rats. Additionally, ET-1 binding in cortex and outer medullary preparations from sl/sl rats was less than one-half that observed in sl/+ rats. Specific binding of ET-1 in the inner medulla was eightfold higher in both sl/sl and sl/+ rats compared with the cortex and outer medulla.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Saturation binding curves were performed using 125I-labeled endothelin (ET)-1 incubated with 20 µg of membrane preparations from sl/+ () or sl/sl () ETB receptor-deficient rat renal cortex (A), outer medulla (B, n = 4-6 animals/group), or 5 µg of inner medulla membrane preparations (C, n = 3 animals/group). Values are means of nonspecific binding subtracted from total binding ± SE.

Scatchard analysis of [¹²⁵I]ET-1 saturation binding curves. To visualize B_max and dissociation constant (K_d) differences between sl/+ and sl/sl animals, Scatchard analyses were performed on nonlinear regression results from [¹²⁵I]ET-1 saturation binding curves (Table 1). Scatchard analyses from both sl/+ and sl/sl renal cortex and outer medulla were linear (data not shown), revealing a one-site model of receptor binding, which was expected. Figure 2 depicts Scatchard plots of inner medullary saturation binding curves of radiolabeled ET-1. Figure 2A shows inner medullary binding results from the sl/+ rat that are linear, indicating a single binding site. However, the Scatchard plot of [¹²⁵I]ET-1 for inner medullary tissue from sl/sl rats (Fig. 2B) reveals a two-site model of receptor binding, each with a distinct K_d. There appears to be one site that has very high affinity (0.08 ± 0.03 nM) but relatively few binding sites (14.2 ± 2.8 fmol/mg protein), whereas the other has lower affinity (5.72 ± 0.79 nM) but is greater in abundance (347.5 ± 25.4 fmol/mg protein).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Scatchard analysis of sl/+ (A, ) and sl/sl (B, ) inner medulla [125I]ET-1 binding curve results.

Saturation binding of [¹²⁵I]ET-3 in renal tissue. Figure 3 represents saturation binding curves using [¹²⁵I]ET-3 incubated with either renal cortex (Fig. 3A), renal outer medullary (Fig. 3B), or renal inner medullary (Fig. 3C) membrane preparations. Specific binding of [¹²⁵I]ET-3 was similar and saturable in the cortex and outer medulla of the sl/+ ET_B-deficient rats. The inner medulla of the sl/+ ET_B-deficient rat exhibited an almost 10-fold greater binding of [¹²⁵I]ET-3 compared with the cortex and outer medulla, indicating a greater number of ET_B receptors in this renal section. Predictably, there was no detectable [¹²⁵I]ET-3 specific binding in the renal cortex and outer medulla from sl/sl ET_B-deficient rats. However, within the renal inner medulla of the sl/sl ET_B-deficient rat, there was an unexpected [¹²⁵I]ET-3 binding.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Saturation binding curves were performed using [125I]ET-3 incubated with 20 µg membrane preparations from sl/+ () or sl/sl () ETB receptor-deficient rat renal cortex (A), outer medulla (B, n = 4-6 animals/group), or 0.5 µg inner medulla membrane preparations (C, n = 3 animals/group). Values are means of nonspecific binding subtracted from total binding ± SE.

B_max and K_d values. By definition, ET-1 binds both ET_A and ET_B receptors, and ET-3 is selective for ET_B receptors at low concentrations; B_max values for [¹²⁵I]ET-3 binding were subtracted from [¹²⁵I]ET-1 B_max values to determine ET_A receptor number in each of the renal sections from sl/+ and sl/sl ET_B-deficient rats. [¹²⁵I]ET-3 receptor binding was used to determine ET_B receptor number. Figure 4 represents B_max values from renal cortical (Fig. 4A), outer medullary (Fig. 4B), and inner medullary (Fig. 4C) tissue from both sl/+ and sl/sl membrane preparations. ET_A receptor number was significantly lower (P < 0.001) in sl/sl cortical (19.95 ± 0.61 fmol/mg protein) and outer medullary (23.75 ± 0.36 fmol/mg protein) membrane preparations compared with the sl/+ cortex (33.40 ± 1.20 fmol/mg protein) and outer medulla (41.49 ± 1.30 fmol/mg protein) preparations. Additionally, there were no detectable ET_B receptors in the sl/sl cortex or outer medulla. ET_A receptor number was not significantly different in the inner medulla between sl/+ and sl/sl animals (60.20 ± 39.89 and 76.00 ± 50.31 fmol/mg protein, respectively). There was a significantly (P < 0.05) lower number of ET_B receptors in the sl/sl inner medulla (211.50 ± 25.36 fmol/mg protein) compared with the sl/+ inner medulla (309.40 ± 26.58 fmol/mg protein); however, the observation that significant [¹²⁵I]ET-3 binding was detected in the inner medulla of sl/sl ET_B receptor-deficient rats was surprising.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Because the ETB receptor has similar affinity for both ET-1 and ET-3, whereas ETA receptors preferentially bind ET-1, [125I]ET-3 receptor binding was used to determine ETB receptor number. Maximal binding (Bmax) values for [125I]ET-3 were subtracted from [125I]ET-1 Bmax values to determine ETA receptor number. ETA receptor number () and ETB receptor number () were calculated for sl/+ and sl/sl renal cortex (A), outer medulla (B), and inner medulla (C). Values represent the means ± SE of Bmax calculations from 3-6 animals/group. Statistical analysis was performed using Student's t-tests with * P < 0.05 and ** P < 0.001.

Binding of [¹²⁵I]ET-1 in whole kidney. Experiments were also performed to explore the possibility that the use of whole kidney homogenate may mask or prevent the detection of ET-3 binding sites in the inner medulla of sl/sl ET_B-deficient rats. Figure 5 shows [¹²⁵I]ET-1 specific binding using inner medulla and whole kidney membrane preparations from either sl/+ or sl/sl ET_B-deficient rats. [¹²⁵I]ET-1 specific binding in the inner medulla of sl/+ ET_B-deficient rats was not significantly different compared with [¹²⁵I]ET-1 specific binding in the whole kidney [1,356 ± 200 and 1,447 ± 354 counts · min¹ (cpm) · µg protein¹, respectively]. However, when inner medullary membrane preparations from sl/sl ET_B-deficient rats were incubated with [¹²⁵I]ET-1, significantly more specific binding occurred compared with that observed in whole kidney membrane preparations (832 ± 74 vs. 72 ± 38 cpm/µg protein, respectively).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Specific binding [counts/min (cpm)] of [125I]ET-1 to either 5 µg inner medullary membrane preparations (n = 3 animals/group) or 90 µg whole kidney membrane preparations (n = 3 animals/group) from sl/+ and sl/sl ETB-deficient rats. Values are means of nonspecific binding subtracted from total binding ± SE. Statistical analysis was performed using Student's t-tests with ** P < 0.001.

Competition binding curves with selective ET_A and ET_B receptor antagonists. Competitive binding experiments were conducted to ascertain whether the novel ET-3 binding in the sl/sl inner medulla could be inhibited by ET_A or ET_B receptor antagonists. Competition binding curves were generated using various concentrations of the ET_A receptor selective antagonist A-127722, the ET_B receptor selective antagonist A-192621, or unlabeled ET-1. Results previously reported using Chinese hamster ovary cells stably transfected with either the ET_A or ET_B receptor report IC₅₀ values using A-127722 as 0.09 nM for the ET_A receptor and 128 nM for the ET_B receptor, whereas the IC₅₀ of ET-1 for the ET_A receptor is 0.28 and 0.14 nM for the ET_B receptor (31). Results from von Geldern and colleagues (30a) report that the ET_B receptor selective antagonist has an IC₅₀ of 1.10 µM for the ET_A receptor and 0.7 nM for ET_B receptor. Thus, in sl/+ inner medullary membrane preparations (Fig. 6A), ET-1 was found to have the highest affinity (IC₅₀ 0.16 ± 0.24 nM; Table 2) followed by the ET_B receptor antagonist A-192621 (IC₅₀ 0.12 ± 0.05 µM; Table 2), whereas A-127722, the ET_A receptor antagonist, had the lowest affinity (IC₅₀ 7.3 ± 0.11 µM; Table 2). However, competition binding curves performed on inner medullary membrane preparations from sl/sl rats were quite different (Fig. 6B). The ET_B receptor antagonist A-192621 displaced the [¹²⁵I]ET-1 with similar concentrations in the sl/sl rats (IC₅₀ 0.11 ± 0.15 µM; Table 2) as it did in the sl/+ animals. Additionally, ET-1 was less able to compete for binding sites as [¹²⁵I]ET-1 (IC₅₀ 1.8 ± 0.14 nM; Table 2), and most strikingly the ET_A receptor antagonist was extremely potent, with an IC₅₀ of 1.2 ± 0.15 pM (Table 2). Additional competition binding experiments performed on Sprague-Dawley inner medullary tissue preparations revealed that the IC₅₀ for ET-1 was 0.18 ± 0.16 nM (Table 2), similar to that of the sl/+ rat. The ET_B receptor antagonist was next highest in affinity for the receptor (IC₅₀ 0.10 ± 0.04 µM; Table 2), again similar to results from the sl/+ rat, whereas the ET_A receptor antagonist had the least affinity of the three ligands, with an IC₅₀ value of 20.7 ± 0.69 µM (Table 2). Taken together, results from the saturation binding curves suggest that there is an ET-3 binding site in the inner medulla of sl/sl rats and that competition binding curves suggest that this site binds the ET_A receptor antagonist with very high affinity.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Competition binding curves were generated from sl/+ (A) and sl/sl (B) ETB-deficient inner medullary membrane preparations (5 µg) incubated with [125I]ET-1 along with various concentrations of either ET-1 (), the ETA receptor antagonist A-127722 (), or the ETB receptor antagonist A-192621 (). Values represent means ± SE (n = 3 animals).

	DISCUSSION

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The present study was conducted to determine if any changes in ET_A receptor binding in the kidney would occur under conditions of ET_B receptor deficiency that is associated with chronic elevations in circulating ET-1 levels. We hypothesized that long-term exposure to ET-1 would cause a downregulation of ET_A receptor expression. Indeed, results showed that ET_A receptor binding was reduced significantly in renal tissue of sl/sl compared with sl/+ rats. These observations are consistent with a compensatory change in ET_A receptor expression when ET_B receptor function is lost and plasma levels of ET-1 are increased. An unexpected observation was evidence for a novel ET-3 binding site in the renal inner medulla of ET_B receptor-deficient animals.

Saturation binding curves using radiolabeled ET-1 reveal an expected decrease in overall ligand-receptor binding interactions in all three regions of kidneys from sl/sl compared with sl/+ rats. Consistent with previous studies, B_max values determined from ET-1 binding were ~10-fold greater in the inner medulla compared with the cortex and outer medulla of both sl/+ and sl/sl rats, highlighting the abundance of ET receptors in this region (16). However, when ET-1 saturation binding results were plotted using Scatchard analysis, the inner medulla from sl/sl rats revealed a surprising two-site model, whereas a linear one-site model was detected in the sl/+ inner medulla. This two-site model found in the sl/sl rat indicated that there was another ET binding site that was different from both ET_A and ET_B receptors. When the results from both the ET-1 and ET-3 saturation curves were plotted as a function of receptor number, the total amount of ET receptors was significantly greater in the inner medulla of both sl/+ and sl/sl animals compared with their respective cortex and outer medulla, with the majority of receptors representing the ET_B subtype. However, B_max values were substantially lower in both sl/+ and sl/sl rats compared with published values from Sprague-Dawley rats (24). The reason for this difference may be related to methodology but could be because of differences in rat strain that have yet to be discerned.

We performed additional experiments to compare previously published ET-1 binding data using whole kidney homogenates with similar whole kidney preparations from sl/+ and sl/sl rats (11). When sl/+ whole kidney or inner medulla membranes were used, ligand-receptor binding was similar between the two preparations. However, the same experiment performed on sl/sl preparations indicates that significant ET-1 binding occurs only when the inner medulla was separated from the cortex and outer medulla, and this binding is "diluted out" when included in a whole kidney preparation. This led us to examine the nature of this binding site through competition binding curves using specific ET_A and ET_B receptor antagonists. Findings from competition binding curves using radiolabeled ET-1 in sl/+ inner medullary preparations indicated that the ET_B receptor antagonist had less affinity compared with the natural ligand, ET-1 but more affinity for the receptor than the ET_A receptor antagonist. However, within the sl/sl inner medulla, the ET_A receptor antagonist had very high affinity, greater than either ET-1 or the ET_B antagonist.

The sl/sl animal is devoid of ET_B receptor apart from adrenergic tissue where its expression in these regions is essential for survival (9). Because plasma ET-1 levels are elevated, these animals have had to adapt to the loss of renal ET_B receptor function in the face of high circulating ET-1. Plasma ET-1 levels in sl/sl rats are significantly elevated compared with sl/+ rats, and results from this colony of rats have been published previously (25). Consistent with our original hypothesis, the sl/sl rat appears to downregulate ET_A receptors in renal cortical and outer medullary tissue. However, the situation in the renal inner medulla appears more complex. Within the inner medulla, there is not a downregulation of ET_A receptors. In fact, ET_A receptor number is similar to those found in the sl/+ inner medulla. Additionally, our studies provide evidence for the existence of a unique binding site that has the novel characteristics of having a high affinity for ET-3 that competes with an ET_A receptor antagonist. We can only speculate, at this point, that this novel binding site represents some form of compensation for the lack of functional ET_B receptors in this region of the kidney. More exhaustive biochemical studies will be needed to determine why this novel binding site has a different binding profile from the traditional ET_A or ET_B receptor. Further studies exploring the possibility that this novel binding site is actually a putative ET receptor along with its estimated size, biochemical behavior, and its pharmacological profile against multiple ET-related ligands are clearly warranted. In addition, to determine whether this binding site is unique to the renal inner medulla, receptor binding experiments will need to be performed using tissue from the sl/sl animals that normally are enriched with ET_B receptors, such as the lungs and the cerebellum.

The physiological significance of this novel ET binding site has yet to be determined, but we can speculate that it could be of importance in models of hypertension that have elevated circulating ET levels. In the DOCA-salt rat model of hypertension, it has been shown that plasma ET-1 levels are elevated compared with placebo-treated controls (1). Additionally, it has been shown that there is an upregulation of ET_B receptors in the renal medulla from DOCA-salt rats whose function is thought to promote sodium and water excretion (27). Thus the DOCA-salt model of hypertension is associated with elevated ET-1 levels, and the kidney responds by upregulating renal medullary ET_B receptors. In rats that lack functional ET_B receptors, there are also elevated plasma ET-1 levels, and these animals have elevated blood pressures (10); we would expect the kidney to respond by upregulating medullary ET_B receptors. However, because there is a mutation in the ET_B receptor gene, we speculate that the sl/sl animal tries to compensate by expressing a renal inner medullary ET binding site that has a unique binding profile different from that of the ET_A and ET_B receptors. We can speculate further that it may influence blood pressure because of its ET_A receptor-like sensitivity to the ET_A antagonist but might also function to promote natriuresis because of its ET_B receptor-like binding of ET-3. Future studies are required to determine if this renal inner medullary binding site has functional significance under normal physiological conditions.

There is anecdotal evidence for the existence of a third class of ET receptors, named ET_C. By definition, the ET_C receptor has a rank order of affinity of ET-3 > ET-1 in radioligand binding and functional studies. In 1993, Karne et al. (17) cloned an ET_C-like receptor that has high affinity for ET-3 from Xenopus dermal melanophores. However, no ET_C receptor has yet been cloned from mammalian sources (17). The possibility arises that the ET-3 binding site is unmasked in the renal inner medulla of the sl/sl ET_B-deficient animal and could be the mammalian counterpart to the ET_C receptor found in Xenopus melanophores. In addition, Zeng and colleagues (34) have cloned and characterized a novel ET receptor type B-like gene enriched in the brain that encodes an ET receptor type B-like protein. This could explain the ET-3 binding found in the inner medulla of the sl/sl animals. Additionally, there is the possibility of a splice variant of this ET receptor type-B-like gene or even a splice variant of the ET_A receptor.

In recent years, evidence has accumulated supporting the notion of alternative splice variants of the ET_B receptor. Cheng et al. (4) identified a splice variant of the ET_B receptor in rat brain. More recently, Nambi and colleagues (24) demonstrated the existence of a splice variant of the ET_B receptor in porcine cerebellum that differs in the last 32 amino acids of the COOH-terminal tail from that of the wild-type receptor but that binding and stimulation of inositol phosphate production are identical to that found in the wild type. Radioligand binding data also indicated that the splice variant and the wild-type ET_B receptor are expressed at the same level in the porcine brain. Additionally, Tsutsumi et al. (30) provide evidence of a novel ET_B receptor transcript that could potentially generate a new receptor. It should be noted that the dopamine--hydroxylase ET_B receptor transgene was constructed using the ET_B cDNA, that is, it does not contain ET_B gene introns, making splice variants from the transgene unlikely.

The sl/sl ET_B-deficient animal has a 301-bp deletion in the gene encoding for the first two membrane-spanning regions of the receptor, and this shortened transcript does not encode functional ET_B receptors (11), normally, the part of the NH₂-terminal domain that is in close proximity to the first transmembrane region required for ligand binding. Through proteolytic truncation experiments, it has been shown that ET-1 binds to a 29-amino-acid sequence in the NH₂-terminal region of the ET_B receptor near the first transmembrane-spanning domain (18, 31). Thus we can speculate that ET-3 binding in the sl/sl inner medulla must be to an ET-like receptor that contains an NH₂-terminal region of 29 amino acids near the first transmembrane segment. Future studies will need to be completed to address whether this binding site has an alteration at the COOH-terminal region of the receptor.

In summary, our data suggest that there is a reduction in ET_A receptor number in the cortex and outer medulla of rats deficient of functional ET_B receptor consistent with a downregulation related to elevated circulating ET-1 levels. Furthermore, we provide evidence for an ET-3 binding site within the inner medulla of ET_B receptor-deficient rats that binds the ET_A antagonist, A-127722, with very high affinity. We speculate that this binding site is a novel ET receptor that is unmasked during ET_B receptor deficiency.