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CARDIOVASCULAR

Identification of -1L Adrenoceptor in Rabbit Ear Artery

Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Matsuoka, Fukui, Japan

Received February 13, 2004; accepted April 20, 2004.

	Abstract

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The -1L adrenoceptor (AR) was identified in rabbit ear artery by both functional and ligand binding studies. In functional studies using arterial rings, the contractile response to NS-49 [(R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluorometh-anesulfonanilide hydrochloride] (-1A and -1L AR-selective agonist) was competitively antagonized with low affinities by prazosin, RS-17053 [N-[2-(2-cyclopropylmethoxyphenoxy) ethyl]-5-chloro-,-dimethyl-1H-indole-3-ethamine hydrochloride], and 5-methylurapidil but with high affinities by tamsulosin and KMD-3213 [(-)-1-(3-hydroxypropyl)-5-[(2R)-2-({2-[(2,2,2-trifluoroethoxy)phenoxy]ethyl}amino)propyl]-2,3-dihydro-1H-indole-7-carboxamide]. In contrast, the response to noradrenaline (nonselective -1 AR agonist) was inhibited noncompetitively by these antagonists (except 5-methylurapidil) with Schild slopes different from unity. These results suggest that the response to NS-49 was mediated predominantly via -1L ARs, whereas the response to noradrenaline was produced through two distinct -1 AR subtypes (presumably -1B and -1L ARs). In binding studies with intact segments of rabbit ear artery, [³H]KMD-3213 bound with high affinity (pK_D = 9.7) to -1 ARs, which were subdivided by prazosin, RS-17053, and 5-methylurapidil into two subtypes (-1A and -1L ARs). In contrast, [³H]prazosin binding sites in ear artery segments (pK_D = 9.8) were identified as -1A and -1B ARs. In conventional binding studies using isolated rabbit ear artery microsomal membranes, [³H]KMD-3213 binding sites were identified as -1A ARs with high affinities for prazosin, RS-17053, and 5-methylurapidil. Our study indicates that an -1L AR having a unique pharmacological profile coexists with -1A and -1B ARs in rabbit ear artery and can be identified either functionally or by binding studies using intact tissues but not microsomal membrane preparations.

View this table: [in this window] [in a new window]   TABLE 1 Functional affinities of -1 AR antagonists estimated in the contractile responses to noradrenaline and NS-49 of rabbit ear artery Subtype selectivity, -1AR subtype selectivity was listed. A, B, D, and L represent -1A, -1B, -1D, and -1L AR subtypes, respectively. Slope, 95% CL: Schild slope and 95% confidence limits were listed. Data represent mean ± S.E.M. of 4 to 12 experiments.

The -1L AR has been detected in various tissues, such as rabbit thoracic aorta (Oshita et al., 1993), rat and human vas deferens (Ohmura et al., 1992; Amobi et al., 2002), rabbit iris (Nakamura et al., 1999), and in the rabbit, rat, and human prostate (Muramatsu et al., 1994; Hiraoka et al., 1995, 1999; Ford et al., 1996). These -1L ARs have been identified primarily by functional studies but have yet to be characterized by ligand binding studies. Hiraoka et al. (1999) have reported that functionally, the -1 ARs of rat prostate are of the -1L subtype, but only the -1A AR was detected by binding studies using microsomal membranes isolated from this tissue. In rabbit iris, the -1A AR is the major receptor subtype detected by reverse transcription-polymerase chain reaction and by membrane binding studies, but functionally, the -1 AR-triggered contractions of the iris dilator muscle can be seen to be mediated via the -1L AR subtype (Nakamura et al., 1999; Suzuki et al., 2002). Thus, there is an apparent discrepancy between the -1 AR subtypes detected by functional and ligand binding studies.

To characterize the pharmacological profiles of many receptors by radioligand binding criteria, most studies have used tissue-derived microsomal membrane preparations. However, it has been pointed out that tissue homogenization may cause dramatic changes in the membrane receptor environment, possibly leading to changes in receptor binding properties (Bylund and Toews, 1993). Recently, we have reevaluated the use of intact tissue segments for measurements of ligand binding, because intact tissue may reflect better the physiological state of a receptor. This method would avoid changing receptor environment by homogenization and would avoid obtaining a low yield of receptor-bearing membranes after fractionation (Tanaka et al., 2004). We hypothesize that the -1L AR observed in functional studies might be identified in the intact tissue before but not after tissue homogenization. In a preliminary study, we used the intact tissue segment binding method to study ARs in rabbit thoracic aorta, a representative tissue showing diverse -1L AR pharmacology (Oshita et al., 1993) However, in this tissue, the nonspecific binding of the radioligand probe was too high to permit a clear evaluation of the AR subtypes present. Therefore, we have turned to a smaller caliber vessel, the rabbit ear artery, with the expectation that a reduced amount of connective tissue in this preparation might lead to a lower degree of nonspecific binding of radioligand. Using the rabbit ear artery, we compared the -1 ARs characterized functionally by a contractile bioassay with the ARs detected by radioligand binding methods using both tissue segments and isolated membrane preparations. As radioligand probes, we used [³H]KMD-3213 (selective for -1A and -1L ARs) and [³H]prazosin (selective for -1A, -1B, and -1D ARs). The -1 AR-mediated contractile responses in rabbit ear artery were produced by noradrenaline (subtype nonselective) and (R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluoromethanesulfonanilide hydrochloride (NS-49) (selective for -1A and -1L ARs) and were characterized pharmacologically with the use of various -1 AR antagonists.

	Materials and Methods

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Animals and Tissue Isolation. Male albino rabbits (2.0–3.5 kg) were anesthetized by injection of sodium pentobarbital (100 mg/kg) into a marginal ear vein and killed. The ears were removed, and the central ear arteries were carefully isolated. Then, fat and connective tissues were removed from the arteries in a modified Krebs-Henseleit solution (120.7 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 2 mM CaCl₂, 1.2 mM NaHPO₄, 25.5 mM NaHCO₃, and 11.5 mM glucose, pH 7.4) gassed with 95% O₂ and 5% CO₂.

Functional Studies. The ear artery tissue was cut into rings of approximately 2 mm in length, and then the endothelium was removed by gentle rubbing with an enamel-coated stainless steel wire. The rings were suspended in an organ bath containing 5 ml of modified Krebs-Henseleit solution gassed continuously with 95% O₂ and 5% CO₂ at 37°C. Desipramine (0.1 µM), deoxycorticosterone acetate (5 µM), and propranolol (1 µM) were added in the bathing solution to block neural and extraneural uptake of noradrenaline and to block -ARs. The removal of endothelium was confirmed by investigating the nonrelaxing response of acetylcholine. Resting tension of 0.5 g was applied to the ear artery rings. After equilibration for at least 60 min, noradrenaline or NS-49 was added to the organ bath in a cumulative manner to obtain concentration-response curves. Various AR antagonists were added to the organ bath 60 min before and during the evaluation of cumulative concentrationresponse curves for noradrenaline or NS-49. The contractile responses were recorded as changes in isometric force measured using an isometric force transducer (T7–30; Orientec, Tokyo, Japan) and amplifier (AP-621G; Nihon Kohden Co., Ltd., Tokyo, Japan).

Tissue Segment Binding Experiments with Rabbit Ear Artery. Tissue segment binding experiments were performed according to the method described by Tanaka et al. (2004) with minor modifications including a small change in the bicarbonate concentration of the incubation buffer. In the modified Krebs-Henseleit solution, pH 7.4, gassed with 95% O₂ and 5% CO₂, the isolated ear artery was cut lengthwise, and the endothelium was removed by gentle rubbing with filter paper. Then, the tissues were cut into small pieces (approximately 4 mm in length) under a stereoscopic microscope. Usually, 30 pieces of ear artery were prepared from one rabbit and were used for one saturation or competition experiment. Each piece was incubated with [³H]KMD-3213 or [³H]prazosin for 12 h at 4°Cin1ml of Krebs' incubation buffer. The composition of the Krebs' incubation buffer was essentially the same as a modified Krebs-Henseleit solution, except the NaHCO₃ concentration was reduced to 10.5 mM to adjust pH to 7.4 in air. The osmolarity of Krebs' incubation buffer was adjusted by adding NaCl. In binding saturation experiments, concentrations of [³H]KMD-3213 and [³H]prazosin ranging from 50 to 1000 pM and from 30 to 250 pM, respectively, were used. Binding competition experiments were performed at a concentration of 500 pM for [³H]KMD-3213 or 100 pM for [³H]prazosin. After incubation, the pieces were gently blotted and rinsed during vortexing for 1 min with 2 ml of incubation buffer at 4°C. The pieces were then blotted and solubilized in 0.3 M NaOH solution to estimate the radioactivity and protein content. The specific binding was determined by subtracting the amount bound in the presence of 3 µM prazosin for [³H]KMD-3213 binding and 30 µM phentolamine for [³H]prazosin binding from the total radioactivity bound per mg protein. In preliminary studies, the same amount of specific binding sites was observed using a hydrophilic competing ligand, adrenaline. Thus, the specific binding sites measured in the present studies appeared not to reflect a nonspecific uptake of the radioligand into the cells. Experiments were done in duplicate at each concentration of radioligand for a saturation experiment or at each concentration of competing ligand for a binding competition experiment. Radioactivity was measured by liquid scintillation counting using a water-miscible scintillation fluid (ULTIMA GOLD; PerkinElmer Life and Analytical Sciences, Boston, MA). Protein concentrations were assayed according to the method of Bradford (1976) using bovine serum albumin as a standard.

Membrane Binding Experiments with Rabbit Ear Arteries. The isolated rabbit ear arteries were pooled and stored at -80°C before use. The arteries were minced with scissors and homogenized in 40 volumes (v/w) of homogenization buffer (50 mM Tris-HCl, 100 mM NaCl, and 2 mM EDTA, pH 7.4) using a polytron homogenizer (specify setting 8, 5 x 20 s at 4°C). The tissue homogenate was subjected to centrifugation at 1000g for 10 min at 4°C. The supernatant was filtered through four layers of gauze (type I) and then centrifuged at 80,000g for 30 min at 4°C. The resulting pellet was resuspended in ice-cold Tris assay buffer (50 mM Tris-HCl and 1 mM EDTA, pH 7.4) and used for binding experiments in the Tris assay buffer. In addition to this membrane preparation, the ear arteries were also homogenized in the Krebs' incubation buffer used in the tissue segment binding experiments. Upon this homogenization, proteinase inhibitors (Complete, EDTA-free tablet, catalog no. 1 873 580; Roche Diagnostics, Mannheim, Germany) were added. After centrifugation as mentioned above, the microsomal fraction was resuspended in the Krebs' incubation buffer and used for binding experiments.

Binding experiments were conducted using the membrane fractions suspended in Tris assay buffer and in Krebs' incubation buffer, respectively. The membranes prepared from ear arteries of seven to nine rabbits were used for one saturation or competition experiment. The incubation was carried out for 45 min at 30°C in Tris assay buffer or for 4 h at 4°C in Krebs' incubation buffer. In binding saturation experiments, 30 to 1000 pM [³H]KMD-3213 was used. Total incubation volume was 2 ml. In binding competition experiments, the membranes were incubated with 100 pM [³H]KMD-3213 in rabbit ear artery in the absence or presence of unlabeled competing ligands. Reactions were terminated by rapid filtration using a Brandel cell harvester onto Whatman GF/C filters presoaked 0.3% polyethylenimine for 15 min, and the filters were then washed three times with 5 ml of ice-cold washing buffer (50 mM Tris-HCl, pH 7.4) or Krebs' incubation buffer, respectively. The resulting filters were dried, and the trapped radioactivity was quantified by liquid scintillation counting. Nonspecific binding of [³H]KMD-3213 was defined as the binding in the presence of 3 µM prazosin. Experiments were done in duplicate at each concentration of [³H]KMD-3213 for a binding saturation experiment or at each concentration of competing ligand for a binding competition experiment. The protein contents of total homogenates obtained before centrifugation and of the microsomal membrane fractions were determined by the method of Bradford (1976).

Data Analysis. Binding data were analyzed using commercially available software (Graph Pad PRISM, version 3.00; GraphPad Software Inc., San Diego, CA). Briefly the data were first fitted to a one- and then a two-site model, and if the residual sums of squares were statistically less for a two-site fit of the data than for a one-site fit, as determined by an F test comparison, then a two-site model was accepted. p values less than 0.05 were considered significant. Abundance of -1 AR in rabbit ear artery was represented as binding capacity per milligram of total tissue protein (B_max: femtomoles per milligram of total tissue protein in ear artery). That is, in the case of conventional binding experiments with membrane fractions, the proteins in the homogenates before fractionation were measured as total tissue protein. Usually, the protein yield of membrane fractions was 1 mg from 12 to 13 mg of total tissue protein of rabbit ear artery. In the case of intact tissue binding, the tissues were solubilized in 0.3 M NaOH solution, and the total proteins were measured, as mentioned above. In functional studies, antagonist affinity estimates (pK_B values) were obtained by plotting the data according to Arunlakshana and Schild (1959). When the straight lines yielded a slope with unity, the pA₂ value estimated was represented as the pK_B value. When a single concentration of antagonist was tested, the pK_B value was also determined for a single concentration of antagonist by the concentration ratio method (Furchgott, 1972).

Data are represented as the mean ± S.E.M. Results were analyzed for statistical significance using the Student's t test. Probability of less than 0.05 was considered significant.

Drugs. The used drugs and their sources were as follows: [³H]KMD-3213 (specific activity 1.92 TBq/mmol), KMD-3213, and tamsulosin from Kissei Pharmaceutical Co., Ltd. (Matsumoto, Japan); NS-49 from Nippon Shinyaku Co., Ltd. (Kyoto, Japan); [³H]prazosin (specific activity 2978.5 GBq/mmol) from PerkinElmer Life and Analytical Sciences; (-)-phenylephrine hydrochloride, phentolamine hydrochloride, desipramine hydrochloride, and prazosin hydrochloride from Sigma-Aldrich (St. Louis, MO); (±)-propranolol hydrochloride and deoxycorticosterone acetate from Nacalai Tesque (Kyoto, Japan); BMY 7378, RS-17053, 5-methylurapidil, and rauwolscine hydrochloride from Sigma/RBI (Natick, MA); and (-)-adrenaline bitartrate and (-)-noradrenaline hydrogen tartrate monohydrate from Wako Pure Chemicals (Osaka, Japan). Prazosin was dissolved in 50% ethanol and diluted with distilled water in functional experiments and with binding buffer in binding experiments. The stock solutions of KMD-3213, RS-17053, and 5-methylurapidil were prepared with dimethylsulfoxide and then diluted with distilled water in functional experiments and with binding buffer in binding experiments.

	Results

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Functional Studies. Noradrenaline and NS-49 produced concentration-dependent contractions in rabbit ear artery. The effective concentrations of noradrenaline spanned a wide range (0.1 nM–10 µM), and the pD₂ value (7.6 ± 0.1, n = 34) was higher, as compared with those of NS-49 (30 nM–100 µM and 6.5 ± 0.1, n = 31) (Fig. 1). The contractile responses to noradrenaline and NS-49 were inhibited by various -1 AR antagonists. However, some differences were observed for the inhibitory actions on both the responses caused by both agonists. Figure 1 shows representative results for the inhibitory effects of prazosin. Prazosin at 1 and 10 nM effectively inhibited the contractile responses to low concentrations of noradrenaline, suggesting that the contractile responses in this concentration range of noradrenaline are mediated through prazosin-high-sensitive -1 ARs, as demonstrated in rabbit thoracic aorta (Oshita et al., 1993; Muramatsu et al., 1998a). At 100 nM, prazosin produced a parallel rightward shift of the concentration-response curve for noradrenaline (Fig. 1A). On the other hand, the concentration-response curve for NS-49 simply shifted to the right after treatment with high concentrations of prazosin (100 and 1000 nM) (Fig. 1B). The slope of the Schild plot for prazosin deviated significantly from unity for the response to noradrenaline but not to NS-49 (Fig. 2; Table 1). The slopes of the Schild plots for tamsulosin and KMD-3213 were also different from unity for the response to noradrenaline but not for NS-49 (Table 1). These results strongly suggested that, in contrast to the response to NS-49, the contractile response to noradrenaline was mediated through at least two distinct -1 ARs that could be distinguished by the antagonists tested. The pA₂ or pK_B values for prazosin and KMD-3213 were significantly different (p < 0.05) for the responses triggered by noradrenaline and NS-49 (Table 1). In contrast, 5-methylurapidil produced a simple competitive inhibition for the responses to both noradrenaline and NS-49, resulting in a Schild plot slope of unity and a low pK_B value. RS-17053 at 0.1 µM had no inhibitory effect on both contractile responses. Therefore, the effect of a single concentration (1 µM) of RS-17053 was tested, with a low pK_B value being estimated by the concentration ratio method (Furchgott, 1972). BMY 7378 (1 µM) and rauwolscine (1 µM) did not cause any inhibitory effects on the contractile responses to noradrenaline and NS-49.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Effects of prazosin in the concentration-responses for noradrenaline (A) and NS-49 (B) in rabbit ear artery. Maximum contractions induced by noradrenaline or NS-49 in the absence of prazosin were taken as 100% (control). Each point represents the mean and S.E.M. of 5 to 12 experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Schild plots for antagonism of prazosin, tamsulosin and KMD-3213 in the contractile responses to noradrenaline (A) and NS-49 (B). Each point represents the mean and S.E.M. of 4 to 12 experiments.

[³H]KMD-3213 and [³H]Prazosin Binding to Tissue Segments of Rabbit Ear Artery. Since distinct -1 ARs have been considered to be involved in the functional responses of rabbit ear artery to noradrenaline and NS-49, tissue segment binding experiments were done using [³H]KMD-3213 and [³H]prazosin as radioligand probes. Experiments were done using Krebs' incubation buffer, which was essentially identical composition with the Krebs-Henseleit solution used in the functional studies (see Materials and Methods). Figure 3 shows the representative saturation binding curves for [³H]KMD-3213 and [³H]prazosin. The specific binding of [³H]KMD-3213 was approximately 90% to 70% of the total binding at concentrations from 50 to 1000 pM and was saturated at concentrations over 600 pM (Fig. 3A). The binding saturation isotherm revealed that [³H]KMD-3213 met the criteria for binding to a single set of sites. The dissociation constant (pK_D) and maximal binding capacity (B_max) were 9.7 ± 0.1 and 218 ± 4 fmol/mg total tissue protein (n = 5), respectively. [³H]Prazosin also showed a concentration-dependent binding, but the proportion of non-specific binding was significantly higher than that of the specific binding at concentrations higher than 250 pM (Fig. 3B), in agreement with a previous report with rat thoracic aorta (Tanaka et al., 2004). Therefore, analysis of [³H]prazosin binding to rabbit ear artery segments was done for concentrations ranging from 30 to 250 pM, resulting in pK_D of 9.8 ± 0.2 and B_max of 202 ± 39 fmol/mg total tissue protein (n = 5).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Saturation curves of [3H]KMD-3213 (A) and [3H]prazosin (B) binding to the intact tissue segments of rabbit ear artery. The specific binding was determined by subtracting the amount bound in the presence of 3 µM prazosin for [3H]KMD-3213 and 30 µM phentolamine for [3H]prazosin from the total radioactivity bound per milligram protein. Each point represents the mean of duplicate determinations. The figure shows a representative result, and similar results were obtained in four other experiments.

The pharmacological profiles of [³H]KMD-3213 and [³H]prazosin binding sites were examined in binding competition experiments. The results are summarized in Table 2. The binding of 500 pM [³H]KMD-3213 to rabbit ear artery segments was biphasically competed for by prazosin, RS-17053 (Fig. 4A), and 5-methylurapidil. The proportions of high- and low-affinity sites were almost equal (Table 2). Tamsulosin and KMD-3213 showed single high affinities in binding competition experiments. However, BMY 7378 competed for the [³H]KMD-3213 binding with a low affinity (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Binding affinities for various -1 AR antagonists estimated in intact tissue segment binding of rabbit ear artery Tissue segment binding experiments were carried out in Krebs incubation buffer at 4°C. The concentrations of [3H]KMD-3213 and [3H]prazosin used in competition experiments were 500 and 100 pM, respectively. Data represent mean ± S.E.M. of four experiments, in which each experiment was done using intact tissue segments of ear arteries isolated from one rabbit.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Competition experiments of [3H]KMD-3213 (A) and [3H]prazosin (B) binding to rabbit ear artery segments by various -1 AR antagonists. [3H]KMD-3213 (500 pM) and [3H]prazosin (100 pM) were incubated with rabbit ear artery segments in the absence and presence of unlabeled drugs. Each point represents mean ± S.E.M. of four experiments.

[³H]prazosin binding at a concentration of 100 pM was biphasically competed for by KMD-3213, RS-17053 (Fig. 4B), and 5-methylurapidil, suggesting that [³H]prazosin bound to two different -1 AR populations in rabbit ear artery segments. The proportions of high- and low-affinity sites were almost equal (Table 2). Tamsulosin and BMY 7378 competed for [³H]prazosin binding monophasically with high and low affinities, respectively.

[³H]KMD-3213 Binding to Rabbit Ear Artery Membranes. The binding experiments with membrane preparations were done using conventional Tris buffer (Oshita et al., 1993; Yang et al., 1997) and in Krebs' incubation buffer, which was essentially identical with that used for intact tissue segment binding procedure. In both cases, [³H]KMD-3213 bound to the microsomal membrane fractions of rabbit ear arteries with a high affinity (pK_D = approximately 10, Table 3A). The B_max values of [³H]KMD-3213 were 35 to 55 fmol/mg total tissue protein in both binding studies. The amount of total binding was approximately one-fourth of the binding capacity detected using the tissue segment binding assay.

View this table: [in this window] [in a new window]   TABLE 3 Binding of [3H]KMD-3213 in microsomal membrane fractions prepared from rabbit ear artery and binding affinities of various -1 AR antagonists Saturation (A) and competition (B) experiments with [3H]KMD-3213 were conducted in Tris buffer at 30°C or in Krebs' incubation buffer at 4°C (see Materials and Methods). Competition experiments were carried out at 100 pM [3H]KMD-3213. Data represent mean ± S.E.M. of four experiments in which ear arteries isolated from seven to nine rabbits were used to prepare the membranes for each experiment.

Prazosin and RS-17053 inhibited 100 pM [³H]KMD-3213 binding in a monophasic manner with high affinities in the competition experiments with Tris assay buffer (Table 3B). The same results were obtained in Krebs' incubation buffer. Thus, prazosin and RS-17053 did not divide the [³H]KMD-3213 binding sites into two components, in contrast with the results obtained using the tissue segment binding approach. Figure 5 shows competition curves for prazosin, tamsulosin, and RS-17053 in Tris assay buffer. Tamsulosin, 5-methylu-rapidil, and BMY 7378 also competed [³H]KMD-3213 binding to rabbit ear artery membranes monophasically in Tris assay buffer (Table 3B). Other membrane binding experiments were not done because of the limited amount of ear artery tissue available for the preparation of microsomal membranes.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Competition experiments with various -1 AR antagonists against [3H]KMD-3213 binding to membranes prepared from rabbit ear artery. Experiments were done in Tris assay buffer and at 100 pM [3H]KMD-3213. Each point represents mean and S.E.M. of four experiments.

	Discussion

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The present study characterized in parallel the pharmacological profiles of functional -1 ARs of rabbit ear artery along with the ligand binding characteristics of the -1 ARs detected using the intact tissue segment and membrane binding approaches. In the functional study, we used two distinct -1 AR agonists, noradrenaline (subtype nonselective) and NS-49 (selective for -1A and -1L ARs) (Muramatsu et al., 1995b). NS-49 over a concentration range less than 3 logarithm units produced concentration-dependent contractions, which were shifted to the right in a competitive manner after treatment with various -1 AR antagonists. Since the slopes of the Schild plots were not different from unity, the responses to NS-49 appear to be mediated through a single -1 AR subtype. The estimated pK_B values for prazosin (7.9), RS-17053 (6.3), and 5-methylurapidil (7.8) were low, whereas the values for KMD-3213 (9.3) and tamsulosin (9.9) were high. At present, three -1 AR subtypes (-1A, -1B, and -1D) have been cloned, all of which have been shown to have a subnanomolar affinity for prazosin (Hancock, 1995; Muramatsu et al., 1995a). As described in the Introduction and Table 1, the -1A AR shows higher affinities for KMD-3213 (pK_i or pK_B = approximately 10.0), RS-17053 (>8.0), and 5-methylurapidil (approximately 9.0), as compared with -1B and -1D ARs (Ford et al., 1996; Kenny et al., 1997; Honner and Docherty, 1999; Murata et al., 1999). Tamsulosin has comparably high affinities for the three AR subtypes with a slightly lower affinity for the -1B AR (>8.5) (Testa et al., 1997; Muramatsu et al., 1998b). BMY 7378 is known to be selective for the -1D AR (>8.0) compared with its affinities (<7.0) for the -1A and -1B ARs (Goetz et al., 1995). These pharmacological criteria determined for -1A, -1B, and -1D ARs are not in accord with the functional affinities estimated for the NS-49-mediated contractile responses of the rabbit ear artery. Rather, the functional affinities more accurately reflect those of the putative -1L AR, which has been demonstrated to have low affinities for prazosin (approximately 8.0), RS-17053 (7.0), and 5-methylurapidil (8.0) and high affinities for KMD-3213 (10.0) and tamsulosin (10.0) (Muramatsu et al., 1995a, 1998b; Ford et al., 1996; Murata et al., 1999; Amobi et al., 2002).

In contrast, the response to noradrenaline in rabbit ear artery was complex because of the wide range (approximately 5 logarithm units) of concentrations of noradrenaline that were necessary to complete the concentration-response curve. Furthermore, the slopes of the Schild plots for prazosin, tamsulosin, and KMD-3213 deviated from unity. These results suggest that the concentration-response curve for noradrenaline may be due to at least two distinct -1 AR subtypes, as suggested in rabbit thoracic aorta (Oshita et al., 1993). High pA₂ or pK_B values for prazosin and tamsulosin but low affinity for KMD-3213, 5-methylurapidil, RS-17053, and BMY 7378 prove an involvement of -1B AR subtype. Low affinity (pK_B: 7.9 and 7.2) for 5-methylurapidil has also been reported in the contractile responses to phenylephrine and noradrenaline of rabbit ear artery (Fagura et al., 1997; Testa et al., 1997).

Then, we examined the -1 AR subtypes detected in the rabbit ear artery using ligand binding methods. Two different approaches were used for the binding study. The first approach uses intact tissue segments, which permit a detection of binding without changing the receptor environment by homogenization or without a loss in the yield of receptor-bearing membranes upon homogenization and fractionation (Tanaka et al., 2004). [³H]KMD-3213 was used to detect the -1L AR because this ligand has been shown to have a high affinity for not only the -1A AR but also the -1L AR (present functional study; Murata et al., 1999). [³H]KMD-3213 bound monotonically to -1 ARs of rabbit ear artery segments with a high affinity. However, in binding competition experiments, the binding sites were divided into two distinct components having different affinities for RS-17053, prazosin, and 5-methylurapidil. [³H]KMD-3213 binding sites were also highly sensitive not only to KMD-3213 but also to tamsulosin. According to the -1 AR subclassification mentioned above, the present results indicate that [³H]KMD-3213 binding sites in rabbit ear artery segments are composed of -1A and -1L AR subtypes. On the other hand, [³H]prazosin binding sites in rabbit ear artery segments showed high affinity for not only prazosin but also tamsulosin and were separated into two components by KMD-3213, RS-17053, and 5-methylurapidil. It would appear that the high-affinity sites for the later three compounds correspond to -1A subtype but that the low-affinity sites correspond to -1B subtype. Since a low concentration of [³H]prazosin (<250 pM) was used because of elevating proportion of the nonspecific binding at the higher concentrations of [³H]prazosin, it appeared that [³H]prazosin bound to prazosin-high-affinity sites only. Thus, it seems that [³H]KMD-3213 detects -1A and -1L ARs in the intact tissue segments of rabbit ear artery, whereas [³H]prazosin binds selectively to -1A and -1B ARs. Since the abundance (B_max) of binding sites of both radioligands was almost equal and the proportions of each subtype in the binding sites were almost same (Table 2), -1A, -1B, and -1L ARs are estimated to occur in the ratio of approximately 1:1:1 in the rabbit ear artery. These binding results suggest, together with the results in functional studies, that both -1B and -1L AR subtypes are mainly involved in the contractile responses in the rabbit ear artery.

On the other hand, in contrast with the intact tissue segment binding data, [³H]KMD-3213-binding sites in the membrane preparations of rabbit ear artery were identified as a single -1A AR component having high affinities for prazosin, RS-17053, and 5-methylurapidil. -1L ARs could not be detected in the membrane preparations, even though the binding conditions were identical to those used for the intact tissue segment binding procedures. The classical radioligand binding assay using microsomal membrane preparations eliminates some nonspecific binding (Bylund and Toews, 1993; Mackenzie et al., 2000) but may result in a loss of receptors upon homogenization and fractionation (Tanaka et al., 2004). This type of loss of receptor upon homogenization has been suggested to be especially important for small tissues (Colucci et al., 1981; Faber et al., 2001). In the present study as in previous reports, the amount of specific [³H]KMD-3213 binding sites in the microsomal membrane fraction was approximately 25% of that estimated using the intact tissue segment binding procedure. The specific [³H]KMD-3213 binding sites apparently lost upon preparing the membranes after homogenization were not detected either in the pellets obtained after centrifugation at 1000g or in the supernatant fraction after microsomal centrifugation (data not shown). From these results, the disappearance of -1L ARs might be in part accounted for by a substantial loss in the yield of receptor-bearing membranes upon homogenization and fractionation. However, a selective loss of -1L AR, in contrast with the -1A AR, cannot be ruled out. Since tissue homogenization changes receptor environment, binding to receptors in membrane preparations may not reflect the binding to receptors present in intact cells (Bylund and Toews, 1993). It is possible that a selective loss of the -1L AR binding phenotype is related to a conformational modification or the environmental changes caused by homogenization. Further studies will be required to clarify this issue.

Ford and coworkers demonstrated that the human -1A AR expressed in Chinese hamster ovary cells had -1L AR characterization in the whole cell binding assay with [³H]prazosin and in the bioassay measuring inositol phosphate accumulation at 37°C, suggesting that -1L AR may be a physiological phenotype of -1A AR observed in the intact cells (Williams et al., 1996; Ford et al., 1997). However, the present study shows that both -1L and -1A ARs coexist in the intact tissue of rabbit ear artery. Recently, we have found that [³H]KMD-3213 bound to only -1A ARs in the intact segments of rat tail artery (I. Muramatsu, T. Tanaka, F. Suzuki, H. Yamamoto, Y. Hiraizumi-Hiraoka, and S. Morishima, unpublished data), where functional -1A and -1B ARs but not -1L ARs have been demonstrated (Lachnit et al., 1997; Murata et al., 1999; Taki et al., 2004). Therefore, even though -1A and -1L ARs may be derived from the same gene, it is likely that both the subtypes are not necessarily expressed in parallel manner. It is interesting to note that -1L ARs have not been observed ubiquitously in -1A AR-expressing tissues (Muramatsu et al., 1995a). Amobi et al. (2002) recently reported that the adrenergic contractions of human vas deferens were mediated by -1L AR in the longitudinal muscle but by -1A AR in the circular muscle, demonstrating an independent distribution of functional -1A and -1L AR subtypes.

In conclusion, the present study shows that the -1L AR subtype can coexist with -1A and -1B ARs in the rabbit ear artery and that the -1L subtype can be identified as a distinct entity either functionally or by a ligand binding approach using intact tissue but not microsomal membrane preparations. The mechanism that accounts for the existence of the -1L AR subtype, possibly as a pleiotropic form of the -1 AR, remains a most interesting topic for further study.

	Acknowledgements

 
We are particularly indebted to the two expert Journal of Pharmacology and Experimental Therapeutics reviewers of this manuscript for critical comments and very thoughtful suggestions. We also express our great appreciation of Professor M. D. Hollenberg (University of Calgary) for helpful discussions concerning our experimental approach and for scrutiny of our manuscript.

	Footnotes

 
This study was supported in part by a Grant-in-Aid for Scientific Research and the 21st COE Research Program (Medical Science) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from the Smoking Research Foundation of Japan.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.066985.

ABBREVIATIONS: AR, adrenoceptor; CL, confidence limit; KMD-3213, (-)-1-(3-hydroxypropyl)-5-[(2R)-2-({2-[(2,2,2-trifluoroethoxy)phenoxy] ethyl}amino)propyl]-2,3-dihydro-1H-indole-7-carboxamide; RS-17053, N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-,-dimethyl-1H-indole-3-ethamine hydrochloride; BMY 7378, (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride; NS-49, (R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluoromethanesulfonanilide hydrochloride.

Address correspondence to: Ikunobu Muramatsu, Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Matsuoka, Fukui 910-1193, Japan. E-mail: muramatu{at}fmsrsa.fukui-med.ac.jp

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