Endothelin-1 Receptors in Rat Tissues: Characterization by Bosentan, Ambrisentan and CI-1020

Yoshinari Yokoyama; Ayaka Osano; Hideki Hayashi; Kunihiko Itoh; Takashi Okura; Yoshiharu Deguchi; Yoshihiko Ito; Shizuo Yamada

doi:10.1248/bpb.b13-00881

Abstract

The present study aimed to characterize comparatively endothelin-1 (ET-1) receptors in rat tissues by radioligand binding assay using [¹²⁵I]ET-1 and to examine receptor binding after oral administration of bosentan. Significant amount of specific [¹²⁵I]ET-1 binding was detected in the lung, heart, kidney, bladder and cerebral cortex of rats. ET-1, bosentan, ambrisentan, and CI-1020 inhibited specific [¹²⁵I]ET-1 binding in these tissues in a concentration-dependent manner. The Hill coefficients of each agent in the rat lung and cerebral cortex and those of bosentan and ET-1 in the heart, kidney and bladder were close to unity, while the Hill coefficients of ambrisentan and CI-1020 in the heart, kidney and bladder were less than one. The nonlinear least squares regression analysis revealed the presence of high- and low-affinity ET-1 receptor sites in these tissues for ambrisentan and CI-1020. Oral administration of bosentan caused a dose-dependent decrease in specific [¹²⁵I]ET-1 binding in the rat lung, kidney and bladder, suggesting significant binding of the tissue ET-1 receptors in vivo. In conclusion, it has been shown that a significant amount of pharmacologically relevant ET-1 receptors may exist in rat tissues and that ET-1 receptor antagonists such as bosentan at pharmacological doses may exert some pharmacological effects by binding these ET-1 receptors.

Endothelin-1 (ET-1) was isolated from the culture media of porcine aortic endothelial cells as the most potent vasoconstrictive peptide identified to date.¹⁾ ET-1 is synthesized by both vascular and non-vascular smooth muscle cells.²⁾ In addition to producing potent contractions in vascular smooth muscle, ET-1 can also produce contractions in non-vascular smooth muscles including the urinary bladder.^3–8) The biological effects of endothelins are mediated through the specific receptors, ET_A and ET_B.^9,10) Both receptor subtypes of ET_A and ET_B mediate the vasoconstrictor and pressor actions of endothelins. ET-1 receptors have been shown to be present in several non-vascular tissues such as the heart, kidney and bladder,^3–10) but the receptor properties such as the subtype distribution in these tissues have not been characterized simultaneously. Also, the receptor binding of ET-1 receptor antagonists has not been comparatively examined in different tissues.

Bosentan, the first non-peptide endothelin receptor antagonist approved for the treatment of pulmonary arterial hypertension, is a nonselective ET_A and ET_B subtype antagonist,^11–13) while ambrisentan and CI-1020 are relatively selective of the ET_A subtype.^12–14) Ambrisentan has also been approved for the treatment of pulmonary arterial hypertension, and has lower hepatotoxicity and weaker drug interaction.^12,13)

The present study aimed to characterize comparatively ET-1 receptors in five tissues (lung, heart, kidney, bladder, cerebral cortex) of rats by a radioligand binding assay using [¹²⁵I]ET-1 as a selective radioligand of the receptor, and to examine receptor binding in these tissues after oral administration of the clinically used bosentan.

MATERIALS AND METHODS

Materials

[¹²⁵I]ET-1 (human) (specific activity, 2200 Ci/mmol) was purchased from DuPont-New England Nuclear (Boston, MA, U.S.A.). Bosentan was obtained from Actelion (Basel, Switzerland). ET-1 and ambrisentan were purchased from the Peptide Institute (Osaka, Japan). Bovine serum albumin was obtained from Nacalai Tesque (Kyoto, Japan). All other chemicals were of analytical grade and were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Animals

Male Sprague-Dawley rats (250–300 g) at 8–10 weeks of age were purchased from Japan SLC (Shizuoka, Japan). They were housed in the laboratory with free access to food and water and were maintained on a 12-h light-dark cycle in a room with controlled temperature (24±2°C).

Animal care and experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University of Shizuoka.

Tissue Preparation and ET-1 Receptor Assay

The tissue preparation and receptor binding assay using a selective radioligand of the endothelin receptor, [¹²⁵I]ET-1 were performed as previously described.^15,16) Rats were exsanguinated by taking the blood from the descending aorta under temporary anesthesia with diethyl ether, and the tissues were then perfused with cold saline from the aorta. The lung, heart, kidney, bladder and cerebral cortex were dissected, and the tissue was minced with scissors and homogenized by a Kinematica Polytron homogenizer in 19 volumes of cold 50 mM Tris–HCl buffer (pH 7.5). The homogenates were then centrifuged at 40000×g for 20 min. The resulting pellet was finally resuspended in cold buffer for the binding assay. The homogenates of rat tissues were incubated with [¹²⁵I]ET-1 (500 pM) in 50 mM Tris–HCl buffer (pH 7.4, 10 mM MgCl₂, and 0.01% bovine serum albumin) (incubation volume: 0.5 mL). Incubation was performed for 120 min at 25°C. The reaction was terminated by rapid filtration (Cell Harvester, Brandel Co., Gaithersburg, MD, U.S.A.) through Whatman GF/B glass fiber filters, and the filters were then rinsed 3 times with 3 mL of cold buffer. Tissue-bound radioactivity was extracted from the filters by placing them overnight by immersion in scintillation fluid, and radioactivity was determined by a gamma counter at 60% efficiency. The specific binding of [¹²⁵I]ET-1 was determined experimentally from the difference between counts in the absence and presence of 100 nM unlabeled ET-1. All assays were conducted in duplicate. Specific [¹²⁵I]ET-1 binding was measured in the absence and presence of various concentrations of ET-1, bosentan, ambrisentan, and CI-1020 in vitro. Their receptor binding affinities (IC₅₀ values) were estimated. Endothelin receptor binding of bosentan was further characterized by measuring specific [¹²⁵I]ET-1 (500 pM) in the rat tissues dissected periodically (1, 3, and 6 h) after the oral administration of vehicle or bosentan at the pharmacological dose of 100 mg/kg. Bosentan was suspended in 0.5% methyl cellulose for the oral treatment in rats.

The single concentration (500 pM) of [¹²⁵I]ET-1 was used in the current study. In the preliminary experiment, Scatchard analysis of specific [¹²⁵I]ET-1 binding in each tissue of rats indicated the dissociation constant (K_d) of 200–300 pM for [¹²⁵I]ET-1 in the tissues except the lung which showed higher K_d value. Thus, we considered 500 pM [¹²⁵I]ET-1 as a reasonable concentration to measure a reliable value of specific [¹²⁵I]ET-1 binding (ET-1 receptor amount). The higher concentration of this radioligand caused higher nonspecific binding of [¹²⁵I]ET-1, suggesting a significant inaccuracy of [¹²⁵I]ET-1 binding assay.

Data Analysis

The analysis of radioligand binding data were basically performed as previously described by Yamada et al.¹⁷⁾ [¹²⁵I]ET-1 binding data were subjected to non-linear regression analysis using Graph Pad PRISM (ver. 4, Graph Pad Software, San Diego, CA, U.S.A.). The ability of bosentan to inhibit specific [¹²⁵I]ET-1 binding was estimated from the IC₅₀, which was the molar concentration of this agent necessary to displace 50% of specific [¹²⁵I]ET-1 binding. Student’s t-test and a one-way ANOVA, followed by the Bonferroni test for multiple comparisons. All data are expressed as the mean±S.E. Significance was accepted at p<0.05.

RESULTS

Characterization of Bladder ET-1 Receptors in Rat Tissues

The endothelin receptors in rat tissues were characterized using [¹²⁵I]ET-1 as a selective radioligand. Significant amount of specific [¹²⁵I]ET-1 binding at concentrations of 500 pM was observed in tissue (lung, heart, kidney, bladder, and cerebral cortex) homogenates of rats.

Fig. 1. Inhibition of Specific [¹²⁵I]ET-1 Binding to the Lung (▲), Heart (◆), Kidney (▼), Bladder (●) and Cerebral Cortex (■) of Rats by ET-1, Bosentan, Ambrisentan and CI-1020

The inhibition of specific [¹²⁵I]ET-1 binding by these agents was determined by incubating the ligand with four to six different concentrations of each agent. Ordinate, percentage of specific [¹²⁵I]ET-1 binding in the absence of any agents. Each point represents the average (n=4–8).

Table 1. pIC₅₀ and Hill Coefficient (nH) for in Vitro Inhibition by Each Agent of Specific [¹²⁵I]ET-1 Binding in the Lung, Heart, Kidney, Bladder, and Cerebral Cortex of Rats

Tissues	Bosentan (ET_A/ET_B)		ET-1 (ET_A/ET_B)			Ambrisentan (ET_A)			CI-1020 (ET_A)
Tissues	pIC₅₀	nH	pIC₅₀	nH		pIC₅₀		nH	pIC₅₀		nH
Lung	7.2±0.1	1.2±0.2	7.5±0.0	1.5±0.4		6.6±0.2		0.9±0.2	7.2±0.1		0.8±0.1
Heart	8.4±0.1	1.2±0.4	8.0±0.1	1.2±0.2	High	9.6±0.6	(62%)	-	11.0±0.2	(58%)	-
Heart	8.4±0.1	1.2±0.4	8.0±0.1	1.2±0.2	Low	8.0±0.4	(38%)	-	8.4±0.4	(42%)	-
Kidney	8.0±0.2	0.8±0.2	7.6±0.1	1.0±0.4	High	9.4±0.4	(33%)	-	10.1±0.3	(33%)	-
Kidney	8.0±0.2	0.8±0.2	7.6±0.1	1.0±0.4	Low	7.0±0.2	(67%)	-	8.1±0.2	(67%)	-
Bladder	8.3±0.1	0.9±0.3	8.1±0.1	1.4±0.3	High	9.5±0.6	(48%)	-	10.1±0.3	(59%)	-
Bladder	8.3±0.1	0.9±0.3	8.1±0.1	1.4±0.3	Low	7.5±0.4	(52%)	-	7.8±0.5	(41%)	-
Cerebral cortex	7.0±0.1	1.2±0.2	8.0±0.1	1.3±0.3		6.2±0.0		1.1±0.1	7.1±0.1		1.2±0.2

Drug inhibition studies were conducted as described in Fig. 1. The pIC₅₀ values and Hill coefficients (nH) were calculated as described in Materials and Methods. The pIC₅₀ and relative proportions of high- and low-affinity binding sites of the [¹²⁵I]ET-1 for ambrisentan and CI-1020 in the heart, kidney and bladder were estimated by the non-linear regression analysis using Graph Pad PRISM (ver. 4, Graph Pad Software, San Diego, CA, U.S.A.). The values in parentheses represent the relative proportion ratio (%). Values are mean±S.E. of 4 to 8 experiments.

The Hill coefficients for these agents in the rat lung and cerebral cortex were close to unity. Hill coefficients for bosentan and ET-1 in the rat heart, kidney and bladder were close to unity (0.8–1.2, while those for ambrisentan and CI-1020 were less than unity (0.3–0.6), as revealed by its shallow inhibition curves of [¹²⁵I]ET-1 binding (Fig. 1).

Nonlinear least-squares regression analysis suggested the existence of two populations of [¹²⁵I]ET-1 binding sites in the heart, one with high (pIC₅₀=9.6±0.6) and the other with low (pIC₅₀=8.0±0.4) affinity for ambrisentan (Table 1). The relative proportions of high- and low-affinity sites for ambrisentan were 62% and 38%, respectively. Nonlinear least-squares regression analysis showed the existence of high-affinity (pIC₅₀=11.0±0.2) and low-affinity (pIC₅₀=8.4±0.4) [¹²⁵I]ET-1 binding sites for CI-1020, with relative proportions of 58% and 42%, respectively, in the heart.

Nonlinear least-squares regression analysis suggested the existence of two populations of [¹²⁵I]ET-1 binding sites in the kidney, one with high (pIC₅₀=9.4±0.4) and the other with low (pIC₅₀=7.0±0.2) affinity for ambrisentan (Table 1). The relative proportions of high- and low-affinity sites for ambrisentan were 33% and 67%, respectively. Nonlinear least-squares regression analysis showed the existence of high-affinity (pIC₅₀=10.1±0.3) and low-affinity (pIC₅₀=8.1±0.2) [¹²⁵I]ET-1 binding sites for CI-1020, with relative proportions of 33% and 67%, respectively, in the kidney.

Nonlinear least-squares regression analysis suggested the existence of two populations of [¹²⁵I]ET-1 binding sites in the bladder, one with high (pIC₅₀=9.5±0.6) and the other with low (pIC₅₀=7.5±0.4) affinity for ambrisentan (Table 1). The relative proportions of high- and low-affinity sites for ambrisentan were 48% and 52%, respectively. Nonlinear least-squares regression analysis showed the existence of high-affinity (pIC₅₀=10.1±0.3) and low-affinity (pIC₅₀=7.8±0.5) [¹²⁵I]ET-1 binding sites for CI-1020, with relative proportions of 59% and 41%, respectively in the bladder.

Effect of Oral Administration of Bosentan on Specific [¹²⁵I]ET-1 Binding Binding in Rat Tissues

Significant decreases (29.9%, 32.3%, and 26.0%, respectively) of specific [¹²⁵I]ET-1 binding in the rat lung were observed 1, 3, and 6 h after oral administration of bosentan at a dose of 100 mg/kg in rats (Table 2). Similarly, significant decreases (49.0%, 56.2% and 45.9%, respectively) of specific [¹²⁵I]ET-1 binding in the rat kidney were observed 1, 3, and 6 h after the oral administration of bosentan at a dose of 100 mg/kg in rats (Table 2). Significant decrease (21.9% and 16.2%, respectively) of specific [¹²⁵I]ET-1 binding in the rat bladder was observed 1 and 3 after oral administration of bosentan at a dose of 100 mg/kg in rats (Table 2). The receptor binding activity was no longer observed at 6 h. The heart showed a tendency of a decrease of specific [¹²⁵I]ET-1 binding. There was little significant change of specific [¹²⁵I]ET-1 binding in the cerebral cortex of bosentan-treated rats.

Table 2. Effects of Oral Administration of Bosentan on Specific [¹²⁵I]ET-1 Binding in Rat Tissues

Doses	Time (h)	Specific [¹²⁵I]ET-1 binding (fmol/mg protein)
Doses	Time (h)	Lung	Heart	Kidney	Bladder	Cerebral cortex
Control		963±182	95.0±20.3	82.1±21.2	160±66	164±29
Bosentan 100 mg/kg
	1	675±121**	81.4±25.8	41.9±10.7**	125±38**	158±23
		(70.1%)		(51.0%)	(78.1%)
	3	652±142**	72.3±25.2	36.0±15.0**	134±21*	140±47
		(67.7%)		(43.8%)	(83.8%)
	6	713±135*	75.2±15.8	44.4±9.7**	145±43	172±24
		(74.0%)		(54.1%)

The specific [¹²⁵I]ET-1 (500 pM) binding was measured for each tissue of rats at 1, 3, and 6 h after the oral administration of bosentan at the pharmacological dose (100 mg/kg). The values in the parenthesis represent the ratio (%) compared with the control value. Values are mean±S.E. of 3 to 10 rats. Asterisks show a significant difference from the control values: * p<0.05; ** p<0.01.

DISCUSSION

The major findings of this study are that 1) there exists a significant amount of specific [¹²⁵I]ET-1 binding sites in the rat lung, heart, kidney, bladder and cerebral cortex, 2) ET_A and ET_B subtypes may coexist in the heart, kidney and bladder, and 3) bosentan orally administered binds significantly ET-1 receptors in the lung, kidney and bladder of rats. The current study aimed to characterize comparatively ET-1 receptors in rat tissues by a sensitive radioreceptor assay using [¹²⁵I]ET-1. There were significant amounts of specific [¹²⁵I]ET-1 binding in the rat lung, heart, kidney, bladder and cerebral cortex, suggesting the existence of ET-1 receptor sites in these tissues. Furthermore, low concentrations of bosentan, ambrisentan, and CI-1020 displayed high affinity to specific [¹²⁵I]ET-1 binding sites in rat tissues (Table 1). Therefore, specific [¹²⁵I]ET-1 binding in rat tissues exhibited pharmacological specificity that characterized ET-1 receptors. These results confirmed mainly previous pharmacological observations demonstrating functional significance of ET-1 receptors.^{4–8,11–14,16)}

Specific [¹²⁵I]ET-1 binding was then measured in the tissues of rats orally administered bosentan. Such experiments could reveal the in vivo binding characteristics of bosentan at ET-1 receptors under the influence of the pharmacokinetics of this agent.^18,19) In the current study, oral administration of bosentan at pharmacologically relevant doses (100 mg/kg) caused a significant decrease of specific [¹²⁵I]ET-1 binding in the rat lung, kidney and bladder, but not in the heart and cerebral cortex (Table 2). To our knowledge, this finding is the first direct evidence for significant binding of pharmacologically relevant ET-1 receptors in the lung, kidney and bladder after the oral administration of a clinically used ET-1 receptor antagonist, which suggests in vivo pharmacological effects on the physiological function. Since the binding of brain ET-1 after the oral administration of bosentan was little observed, this agent might be poorly permeable through the blood–brain barrier. The reason why oral administration of bosentan displayed little significant binding of ET-1 receptors in the rat heart is not clear at the present time, but it may be due to the low tissue distribution or the rapid dissociation from the receptors of this agent at the current dose in the heart.

ET-1 displays physiological effects by the binding to the specific receptor subtypes of ET_A and ET_B.^9,10) The radioligand binding technique is useful to reveal the relative population of receptor subtypes using selective antagonists.¹⁸ Bosentan is a nonselective ET_A and ET_B subtype antagonist,^11–13) while ambrisentan and CI-1020 are relatively selective of the ET_A subtype.^12–14) In the present study, the competition curve by bosentan, ambrisentan and CI-1020 of specific [¹²⁵I]ET-1 binding in the rat lung and cerebral cortex was monophasic with unity of the Hill coefficient, suggesting no heterogeneity. Bosentan displayed a monophasic competition curve of specific [¹²⁵I]ET-1 binding in each tissue of rats. On the other hand, both ambrisentan and CI-1020 displayed shallow inhibition curves of specific [¹²⁵I]ET-1 binding in the rat heart, kidney and bladder (Fig. 1). The Hill coefficients for both agents-induced competitive [¹²⁵I]ET-1 binding were less than unity, suggesting the heterogeneity of the binding sites or negative cooperativity.

Nonlinear least squares regression analysis revealed the presence of high-affinity and low-affinity ET-1 receptor sites for ambrisentan with relative proportions of 62% and 38%, respectively, in the heart (Table 1). Similar analysis of the biphasic inhibition by CI-1020 in the heart showed the existence of two classes of ET-1 binding sites with distinct affinities for this agent, with the relative proportions of high-affinity (58%) and low-affinity (42%) sites which agreed with those of the respective sites for ambrisentan.

Nonlinear least squares regression analysis revealed the presence of high-affinity and low-affinity ET-1 receptor sites for ambrisentan with relative proportions of 33% and 67%, respectively, in the kidney (Table 1). Similar analysis of the biphasic inhibition by CI-1020 in the kidney showed the existence of two classes of ET-1 binding sites with distinct affinities for this agent, with the relative proportions of high-affinity (33%) and low-affinity (67%) sites which agreed with those of the respective sites for ambrisentan.

Nonlinear least squares regression analysis revealed the presence of high-affinity and low-affinity ET-1 receptor sites for ambrisentan with relative proportions of 48% and 52%, respectively, in the bladder (Table 1). Similar analysis of the biphasic inhibition by CI-1020 in the bladder showed the existence of two classes of ET-1 binding sites with distinct affinities for this agent, with the relative proportions of high-affinity (59%) and low-affinity (41%) sites. Taken together, these results suggest that the rat heart, kidney and bladder may contain both subtypes of the ET_A and ET_B receptors at different ratios.

The current results with radioreceptor binding studies using [¹²⁵I]ET-1 have confirmed previous functional and biochemical studies that ET_A and ET_B receptors coexist in the heart, kidney and bladder of humans and animals.^20–24) The coexistence of ET_A and ET_B in the kidney was recently shown by physiological and pharmacological studies.^25,26) Also, Francis et al.²⁷⁾ showed a differential regulation of ET_A and ET_B receptors in the renal tissue of rats with compensated and decompensated heart failure. ET_B receptors have a critical role in protecting target organs such as the heart. In fact, it was previously shown that ET_B receptor than ET_A receptor may contribute more greatly to the pharmacological effects of bosentan in the human hypertension²⁸⁾ and in rats with pulmonary hypertension.²⁹⁾

Autoradiographical study by Khan et al.^30,31) showed the presence of ET_A and ET_B receptors in the bladder of normal and partial bladder outlet-obstructed rabbits. Ukai et al.³²⁾ showed that a selective ET_A receptor antagonist had ameliorating effects on various urinary dysfunctions, including benign prostatic hyperplasia. These results implicate the functional significance of bladder ET_A and ET_B receptors. Thus, the relative density of ET_A and ET_B receptor subtypes may differ among rat tissues.

Ambrisentan and CI-1020, ET_A-subtype selective antagonists, showed relatively low affinity for specific [¹²⁵I]ET-1 binding sites in the lung. This result may coincide with the previous observation by positron emission tomography using ¹⁸F-labelled endothlein-1 that elucidated the function of ET_B receptor subtype as a clearing receptor in organs expressing high densities including lung and kidney of rats.²⁴⁾ Furthermore, Nagase et al.²⁰⁾ and Hay et al.²¹⁾ revealed the presence of ET_B receptor subtype which may mediate contraction induced by ET-1 or other ET-1 ligands in the airway smooth muscle of mice and in human bronchus. Also, predominant existence of ET_B receptor was shown in the guinea-pig lung by the quantitative autoradiographic study.³³⁾

The present study demonstrated that pharmacologically relevant ET-1 receptors are present in the rat lung, heart, kidney, bladder and cerebral cortex, and that these receptors in the lung, kidney and bladder may be significantly bound after the oral administration of bosentan at the pharmacological dose.

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