Pharmacological analysis of the novel, selective α₁-adrenoceptor antagonist, KMD-3213, and its suitability as a tritiated radioligand

Abstract

1. Pharmacological profiles of tritiated KMD-3213, a new antagonist of α₁-adrenoceptor (AR), were examined in recombinant and native α₁-AR, and compared with those of prazosin (PZ) and tamsulosin (YM-617).

2. In saturation experiments, [³H]-KMD (10–2000 pM) showed high affinity for α_1a-AR (pK_D=10.5). However, no significant binding to α_1b-AR and insufficient/unsaturated binding to α_1d-AR were observed at concentrations up to 2000 pM. In contrast, [³H]-PZ and [³H]-YM bound to all subtypes with high affinity (pK_D>9). In competition experiments, KMD-3213 also had higher affinity for α_1a-AR than for other two subtypes; pK_i=10.4, 8.1 and 8.6 for α_1a-, α_1b- and α_1d-AR, respectively.

3. [³H]-KMD also bound to the native α_1A-AR (rat submaxillary gland) with high affinity, but not to α_1B-AR (rat liver). In rat kidney which expresses α_1A- and α_1B-AR, [³H]-KMD and [³H]-PZ bound to a single high-affinity site (pK_D=10.8 and 10.1, respectively) with distinct amount of binding sites (B_max=159 and 267 fmol mg⁻¹ protein, respectively). [³H]-PZ binding sites consisted of low- and high-affinity sites for KMD-3213 (pK_i=7.6 and 10.7, respectively), for WB4101 (pK_i=8.1 and 10.0) and for YM-617 (pK_i=8.7 and 10.8). The proportion of the high affinity site was approximately 60% in these drugs which was compatible to the ratio between B_max of [³H]-KMD and [³H]-PZ. [³H]-KMD binding sites consisted of a single site for these drugs with affinities which were similar to those of the high affinity sites in [³H]-PZ binding.

4. In functional experiments, KMD-3213 antagonized the contractile responses to NS-49 or noradrenaline (NA) with higher affinity in functional α_1A- (rat caudal artery, pA₂=10.0 against NS-49) and α_1L-AR (dog mesenteric artery, pA₂=9.9 against NA) than in α_1B- (dog carotid artery, pA₂=7.7 against NA) and α_1D-AR (rat thoracic aorta, pA₂=8.3 against NA).

5. These results confirm the α_1A-AR selectivity and high affinity of KMD-3213, and indicate that [³H]-KMD can label selectively α_1A-AR.

Introduction

α₁-Adrenoceptors (ARs) constitute a heterogeneous family of receptors (McGrath, 1982). The existence of two subtypes, α_1A- and α_1B-AR, was initially suggested in pharmacological studies (Han et al., 1987; Minneman, 1988; Morrow & Creese, 1986). Molecular cloning techniques have revealed the existence of at least three receptor subtypes (α_1a-, α_1b- and α_1d-AR), and pharmacological studies indicated that these clones subtypes correspond to native α_1A-, α_1B- and α_1D-AR subtypes, respectively (Bylund et al., 1995; Hieble et al., 1995). The fourth subtype, α_1L-AR which is defined principally based on a distinct low affinity for prazosin (PZ), has been proposed mainly from functional studies (Flavahan & Vanhoutte, 1986; Muramatsu et al., 1990; 1991; Oshita et al., 1991; Ford et al., 1996). However, the molecular identity of α_1L-AR still remains to be clarified (Bylund et al., 1998).

Various α₁-AR ligands are used for characterizing the binding profiles and functions of receptor subtypes; however, few ligands showing subtype selectivity are available. Recently, KMD-3213 [(−)-(R)-1-(3-hydroxypropyl)-5-[2-[[2-[2-(2,2,2-trifluoroethoxy) phenoxy] ethyl] amino]propyl]indoline - 7 - carboxamide] was reported as an α₁-AR antagonist (Shibata et al., 1995). In the present study, we characterized the binding profiles of KMD-3213, and its tritiated form as a radioligand, by using recombinant α₁-AR subtypes and native tissues, and compared α₁-AR subtype selectivity of KMD-3213 with other α₁-AR antagonists such as PZ and tamsulosin (YM-617). The characteristics of KMD-3213 were also compared with those of other drugs in functional experiments.

Methods

Male Wistar rats (250–350 g) and Beagle dogs (10–15 kg) were used for the experiments. Animals were sacrificed under anaesthesia with pentobarbital, and tissues were isolated immediately thereafter.

Membrane preparation

For the cloned ARs, COS-7 cells were transfected with the cDNA clones of human α_1a-, hamster α_1b- and rat α_1d-AR. The harvested cells were suspended in ice-cold assay buffer (Tris-HCl 50 mM, EDTA 1 mM, pH 7.4), sonicated and centrifuged at 3000×g for 10 min. The supernatant was then centrifuged at 80,000×g for 30 min, and the resulting pellet was resuspended in assay buffer and used for binding experiments.

For preparation of native receptor subtypes, isolated rat tissues (submaxillary gland, liver and kidney) were homogenized in 20 vol. of ice-cold homogenization buffer (mM): Tris-HCl 50, NaCl 100, EDTA 2, pH 7.4) with a Polytron (setting 8, 15 s×3) and filtered through four layers of cheese cloth. The supernatants were centrifuged at 80,000×g for 30 min, and the resulting pellets were suspended in ice-cold assay buffer, and then again centrifuged at 80,000×g for 30 min. All centrifugation was done at 4°C. Final pellets were resuspended in assay buffer and used for the following experiments.

Radioligand binding experiments

In saturation binding experiments, the membranes were incubated with various concentrations of [³H]-ligands for 45 min at 30°C. Total incubation volume for [³H]-PZ, [³H]-YM and [³H]-KMD was 1 ml, 3 ml and 2∼3 ml, respectively (5–30 μg protein/tube for cultured cells or 50–150 μg protein/tube for native tissues). Nonspecific binding was defined as binding in the presence of 1 μM YM-617 for [³H]-PZ or [³H]-KMD or in that of 1 μM PZ for [³H]-YM, excepting the binding to rat liver and kidney (0.3 μM YM-617 for [³H]-PZ and 0.3 μM PZ for [³H]-KMD). In competition binding experiments, membranes were incubated with about 200 pM [³H]-PZ or 70∼100 pM [³H]-KMD and unlabelled drugs for 45 min at 30°C. Specific binding of both radioligands (200 pM [³H]-PZ and 100 pM [³H]-KMD) was approximately 90% and 80% of the total binding for cloned cells and native tissues, respectively. Reactions were terminated by rapid filtration with a Brandel cell harvester onto Whatman GF/C filters presoaked in 0.3% polyethyleneimine for 15 min. The filters were then washed four times with 4 ml of ice-cold 50 mM Tris-HCl (pH 7.4) and dried. The filter-bound radioactivity was determined by liquid scintillation counting. Experiments were conducted in duplicate. Binding affinities of [³H]-ligands and unlabelled drugs were expressed as negative logarithm of the equilibrium dissociation constant (pK_D and pK_i, respectively). Protein concentrations were quantified by the method of Bradford using bovine serum albumin as standard (Bradford, 1976).

Functional experiments

Freshly isolated arteries were cleaned of adherent connective tissue and cut helically, and the endoethelium was removed by gentle rubbing. The strip was then mounted vertically in an organ bath containing 20 ml of Krebs-Henseleit solution (composition in mM): NaCl 112.0, KCl 5.9, CaCl₂ 2.0, MgCl₂ 1.2, NaH₂PO₄ 1.2, NaHCO₃ 25.0 and glucose 11.5, gassed with 5% CO₂ and O₂ and maintained at 37°C. Resting tension applied was 0.5 g for rat caudal artery or 1 g for other tissues, and the responses were recorded isometrically through force-displacement transducers. All preparations were equilibrated for 90 min, and the following experiments were performed.

Concentration-response curves were obtained by adding the agonist (NS-49 for rat caudal artery or noradrenaline (NA) for other arteries) in cumulative fashion. The curves were drawn at least five times for the same strip, and the third curve was used as the control. After the control experiment, strips were incubated with α₁-AR antagonists for 30 min before, and during, the construction of the curves. All the experiments were performed in the presence of 0.1 μM desipramine and 5 μM deoxycorticosterone acetate to block neural and extraneural uptake, respectively, of NA and 3 μM propranolol to block β-AR induced responses. In rat caudal artery, 0.1 μM rauwolscine was further applied in addition to those three chemicals to avoid α₂-AR mediated responses.

Data analysis

Data were given as means±s.e.mean. Saturation and competition binding data were first fitted to a one- and then a two-site model, and the two-site model was accepted only if it resulted in a significant improvement of the fit as judged by an F-test comparison with a P<0.05. Analysis of radioligand binding data was performed with LIGAND (Munson & Rodbard, 1980), a nonlinear curve-fitting program.

In functional studies, antagonist affinity estimates were obtained by construction of Schild regressions and were constrained to a slope of unity (if not statistically different) according to the equation: pK_B=log(r-1)−log[antagonist], where r is the concentration ratio between EC₅₀ values (concentration for a half-maximal response) in the presence and absence of the antagonist. Schild plots were also constructed by plotting the log(r-1) against the log of antagonist concentration, and pA₂ values were determined from the intercept (Arunlakshana & Schild, 1959).

Drugs

The drugs used and their sources were following: (−)-(R)-1-(3-hydroxypropyl) -5 - [ 2- [[2 -[2 - (2,2,2 - trifluoroethoxy) phenoxy] -ethyl]amino]propyl]indoline-7-carboxamide (KMD-3213), tamsulosin HCl (YM-617) and (R)-(−)-3′-(2-amino-1-hydroxyethyl)-4′-fluoromethane-sulphonanilide hydrochloride (NS-49), from Kissei Pharmaceutical Co. Ltd. (Matsumoto, Japan); prazosin HCl (PZ), (−)-noradrenaline bitartrate (NA) and desipramine hydrochloride, from Sigma (St. Louis, U.S.A.); WB4101, BMY7378 and rauwolscine hydrochloride, from Research Biochemicals Inc. (Natick, U.S.A.); (±)-propranolol hydrochloride and deoxycorticosterone acetate, from Nacalai (Kyoto, Japan); [³H]KMD-3213 (49∼52 Ci mmol⁻¹), from Amersham (England); and [³H]prazosin (77.2 Ci mmol⁻¹) and [³H]-YM-617 (32.5 Ci mmol⁻¹), from NEN (Boston, U.S.A.).

KMD-3213 was dissolved in dimethylsulphoxide and diluted in assay buffer for binding studies or Hartmann's solution (composition in w v⁻¹%): NaCl 0.60, KCl 0.03, CaCl₂ 0.02 and sodium lacate 0.31, for functional studies. [³H]-ligands were diluted in assay buffer. PZ was dissolved in 50% ethanol and diluted in distilled water. Other drugs were dissolved in and diluted with distilled water.

Results

Characteristics of tritiated KMD-3213

Chemical structures of tritiated KMD-3213, (−)-(R)-1-(3-hydroxypropyl) - 5 - [2 -[[2-[2-(2,2,2-trifluoroethoxy)[4,6(n)-³H]phenoxy]ethyl]amino]propyl]indoline-7-carboxamide, is shown in Figure 1a. This tritiated form of KMD-3213 ([³H]-KMD) with high specific radioactivity (49∼52 Ci mmol⁻¹) effectively bound to α_1a/α_1A-AR at subnanomolar concentrations. A representative saturation curve of [³H]-KMD at human α_1a-AR is shown in Figure 1b. The binding was equilibrated by incubation at 30°C for more than 40 min (data not shown). Non-specific binding was less than 10% at 100 pM [³H]-KMD. Since [³H]-KMD had high affinity for α_1a/α_1A-AR, 2∼3 ml of incubation volume was employed in binding experiments in order to avoid a significant reduction of free [³H]-KMD concentration during incubation.

Figure 1.

Characteristics of [³H]KMD-3213. (a) Chemical structure of [³H]KMD-3213. Asterisks show the tritiated sites. (b) Saturation of [³H]KMD3213 binding to human α_1a-AR subtypes expressed in COS-7 cells. Each point represents the means of duplicate determinations from a single experiment. The experiments were replicated three times with similar results.

Radioligand binding experiments for cloned α₁-AR subtypes

In saturation experiments, specific binding of the three [³H]-ligands ([³H]-PZ, [³H]-YM and [³H]-KMD) was determined by use of the same membranes in each experiment, and representative patterns are shown in Figure 2. All of the specific binding curves were saturable, except the curves for [³H]-KMD binding to α_1b- and α_1d-AR. In contrast to [³H]-PZ and [³H]-YM which showed high affinity for the three cloned α₁-AR subtypes (pK_D>9), [³H]-KMD clearly showed different sensitivity to them; high affinity to α_1a-AR (pK_D; 10.5±0.07), no significant binding to α_1b-AR and insufficient/unsaturated binding to α_1d-AR at concentrations up to 2000 pM (Table 1) were observed.

Figure 2.

Saturation binding experiments of [³H]-PZ, [³H]-YM and [³H]-KMD to recombinant α₁-AR subtypes. (a) Human α_1a-, (b) hamster α_1b- and (c) rat α_1d-AR subtypes were expressed in COS-7 cells. Specific binding, drawn in Figures, was determined as the difference in radioligand binding in the presence or absence of 1 μM YM-617 for [³H]-PZ and [³H]-KMD or 1 μM PZ for [³H]-YM. All three radioligands were incubated with the same membrane in each experiment. A representative result for each subtype is shown as mean of duplicate in a single experiment (see Table 1).

Table 1.

Binding affinity of radioligands for cloned α₁-AR subtypes

Several α₁-AR antagonists were tested in competition experiments against [³H]-PZ or [³H]-KMD binding to cloned α₁-AR subtypes. The competition experiment with [³H]-KMD binding was done only with α_1a-AR, because [³H]-KMD at concentrations used in this experiment did not show sufficient binding to α_1b- and α_1d-AR (Figure 2). The affinities of the α₁-AR antagonists to cloned α₁-AR subtypes are shown in Table 2. The characteristics of each antagonist were basically similar between [³H]-PZ and [³H]-KMD binding. Regarding the selectivity for α₁-AR subtypes, several patterns were observed. PZ showed no selectivity for the three α₁-AR subtypes. YM-617 and WB4101 showed slightly lower affinity for α_1b-AR than for other subtypes, and BMY7378 showed higher affinity for α_1d-AR than for other two subtypes. KMD-3213 had prominent selectivity, showing more than 200 and 50 times higher affinity for α_1a-AR than for α_1b- and α_1d-AR, respectively.

Table 2.

Profile of α₁-AR antagonist binding to cloned α₁-ARs

Binding characteristics for native α₁-AR subtypes

The binding of [³H]-KMD was further characterized in rat submaxillary gland, liver and kidney, which are pharmacologically defined as tissues predominantly containing α_1A-AR, α_1B-AR and a mixture of α_1A- and α_1B-ARs, respectively (Michel et al., 1989; Feng et al., 1991). As shown in Table 3, [³H]-PZ showed high affinity for all tissues, whereas [³H]-KMD bound with high affinity to the submaxillary gland and kidney but showed no saturable binding to liver membranes at concentrations up to 2000 pM (data not shown). B_max values of these two tritiated compounds were similar in submaxillary gland, but in kidney B_max of [³H]-KMD was only 60% of that of [³H]-PZ.

Table 3.

Characteristics of radioligand binding to native tissues

The binding characteristics of series of antagonists for the native α₁-AR subtypes were examined in competition experiments as shown in Table 4. In submaxillary gland (α_1A-AR) and liver (α_1B-AR), the binding of [³H]-PZ and [³H]-KMD was displaced monophasically by the competitors used with affinities which were compatible with those shown in Table 2. In rat kidney, however, YM-617, KMD-3213 and WB4101 displaced the binding of [³H]-PZ with two distinct affinity sites (Figure 3). The affinities for the high and low affinity sites corresponded well to those for α_1A- and α_1B-AR subtype, respectively. The proportion of high affinity sites in the total binding of [³H]-PZ were 68, 58 and 66% for YM-617, KMD-3213 and WB4101, respectively, which matched well with the ratio (60%) between B_max values of [³H]-KMD and [³H]-PZ seen in Tables 3 and 4. The binding of [³H]-KMD was uniformly displaced by a series of drugs with affinities corresponding to those for high affinity sites in the displacement of [³H]-PZ binding (Figure 3 and Table 4). These results suggest that [³H]-KMD recognized a single population of binding sites (α_1A-AR) from the mixed populations (α_1A- and α_1B-AR) of binding sites in rat kidney because of its high affinity for α_1A-AR.

Table 4.

Binding affinity of α₁-AR antagonists to native tissues

Figure 3.

Competition experiments of specific [³H]-PZ and [³H]-KMD binding by various α₁-AR antagonists in rat kidney. (a) 200 pM [³H]-PZ or (b) 100 pM [³H]-KMD was incubated with rat kidney membranes in the presence of unlabelled drugs. Competition curves of YM-617, KMD-3213 and WB4101 against [³H]-PZ binding were significantly fitted to two-site model (P<0.05), and other curves were fitted to one-site model. Data are shown as mean±s.e.mean of three independent experiments.

Functional experiments in isolated native tissues

To further characterize the selectivity of KMD-3213, we performed functional experiments using isolated rat caudal arteries, dog carotid arteries, rat thoracic aortae and dog mesenteric arteries in which agonist-induced contraction is mediated by α_1A-, α_1B-, α_1D- and α_1L-AR subtype, respectively (Lachnit et al., 1997; Muramatsu et al., 1990; 1991; Kenny et al., 1995). In rat caudal artery, the contractile response to NA was mediated by at least two α₁-AR subtypes; one is α_1A-AR and the other remains to be defined (Lachnit et al., 1997). We employed NS-49, an α_1A-AR selective agonist (Obika et al., 1995), to draw the response mediated by α_1A-AR in rat caudal artery. In other tissues NA was employed as an agonist. The representative antagonism of KMD-3213 are shown in Figures 4 and 5, and the results are summarized in Table 5. All of the antagonists shifted the agonist-induced concentration-response curves to the right in a parallel fashion, and Schild analysis revealed that slopes of all the antagonists in any tissues were close to unity (Figures 4 and 5, Table 5). We, thus, concluded that the antagonist was fully equilibrated under these conditions. PZ displayed high affinity for α_1A-, α_1B- and α_1D-AR (pA₂ >9), but low affinity for α_1L-AR. In contrast to PZ, KMD-3213 showed higher affinity for functional α_1A- and α_1L-AR than for α_1B- and α_1D-AR displaying pA₂ values of 10.0, 9.9, 7.7 and 8.3, respectively. The ratios of affinity of KMD-3213 for α₁-AR subtypes are approximately 200 for α_1B/(α_1A or α_1L) and 50 for α_1D/(α_1A or α_1L), which are similar values with those obtained in binding experiments. YM-617 showed high affinity for all subtypes (pA₂ >9), and BMY7378 displayed α_1D-AR selectivity.

Figure 4.

Antagonism of contractions to agonists by KMD-3213. (a) Concentration response curves to NS-49 in rat caudal artery are shown. •, Control; ▴, KMD-3213 0.03 nM; ⧫, 0.3 nM; ▪, 3 nM. (b) Concentration response curves to NA in dog carotid artery are shown. •, Control; ▴, KMD-3213 0.03 μM; ⧫, 0.3 μM; ▪, 3 μM. Data are shown as mean±s.e.mean of 5–6 independent experiments.

Figure 5.

Comparison of Schild analyses for KMD-3213 and YM-617. Schild plots constructed with data from this Figure and Table 5 for KMD-3213 (a) or YM-617 (b) against contractile responses to NS-49 in rat caudal artery □, or to NA in dog carotid artery ▵, in rat thoracic aorta ○, and in dog mesenteric artery •. Data are shown as mean±s.e.mean of 5–6 independent experiments

Table 5.

Functional affinity of α₁-AR antagonists for native tissues

Discussion

KMD-3213, a recently synthesized α_1A-AR antagonist, displays antagonism with apparent tissue selectivity (Moriyama et al., 1997; Yamagishi et al., 1996). In the present study, we characterized the pharmacological profiles of KMD-3213 by use of its tritiated form as a radioligand and compared it with those of other α₁-AR ligands, [³H]-PZ and [³H]-YM.

At first, we compared three α₁-AR specific radioligands, [³H]-PZ, [³H]-YM and [³H]-KMD, in saturation binding experiments with recombinant α_1a-, α_1b- and α_1d-AR subtypes. [³H]-PZ showed high affinity for all α₁-AR subtypes without apparent subtype selectivity. [³H]-YM binding had slightly less affinity at α_1b-AR than at α_1a- and α_1d-ARs, however, relatively high affinity was observed for all subtypes as has been described previously (Han et al., 1995). In contrast, [³H]-KMD showed high affinity for the α_1a-AR subtype only; [³H]-KMD binding to α_1b- and α_1d-AR subtypes was insufficient and not saturated at concentrations up to 2000 pM (Table 1 and Figure 2). This high selectivity of KMD-3213 to α_1a-AR was also confirmed in competition binding experiments. Although YM-617 and WB4101 also showed higher affinity for the α_1a-AR subtype, the degree of selectivity was less than that of KMD-3213 (Table 2). These results were in good agreement with results of a previous report (Shibata et al., 1995), in which three recombinant subtypes of human were investigated with unlabelled KMD-3213. This suggests that the selectivity of KMD-3213 is not dependent on species and indicates that [³H]-KMD could distinguish the α_1a-AR subtype from the other two recombinant subtypes.

At native α₁-AR subtypes, [³H]-KMD showed similar subtype selectivity, i.e. high affinity for α_1A-AR of rat submaxillary gland and no significant binding to α_1B-AR of rat liver (Table 3). This selectivity was also applied to rat kidney which contains both α_1A- and α_1B-AR (Feng et al., 1991). Both [³H]-PZ and [³H]-KMD binding revealed single populations of binding sites with high affinity but showed different densities; B_max of [³H]-KMD was 60% of that of [³H]-PZ. In addition, the binding of [³H]-PZ consisted of two components with high- (α_1A-AR) and low- (α_1B-AR) affinity sites discriminated by YM-617, KMD-3213 and WB4101 (Figure 3 and Table 4). The proportion of high affinity sites in the total binding sites of [³H]-PZ were 68, 58 and 66% for YM-617, KMD-3213 and WB4101, respectively, which matched well with the ratio (60%) between the B_max values of the two [³H]-ligands (Table 4). In contrast, the binding of [³H]-KMD was displaced by these drugs monophasically with affinity estimates which correspond well with those for α_1A-AR. These results demonstrated that [³H]-KMD exclusively detected an α_1A-AR population from the mixture labelled by ³H-PZ. Since it is impossible to investigate the binding characters of α_1D-AR in native tissues (Yang et al., 1997), we conducted further functional experiments.

Rat caudal artery, dog carotid artery, rat thoracic aorta and dog mesenteric artery were employed in functional experiment as tissues in which agonist-induced contraction is mediated by α_1A-, α_1B-, α_1D- and α_1L-AR subtype, respectively (Lachnit et al., 1997; Muramatsu et al., 1990; 1991; Kenny et al., 1995). The α_1L-AR, the fourth subtype (Bylund et al., 1998), has been proposed based on its distinct low affinity for PZ mainly in functional experiments (Flavahan & Vanhoutte, 1986; Muramatsu et al., 1990; Ohmura et al., 1992). This α_1L-AR has not yet been identified separately as a distinct gene product and is therefore not fully characterized. Recently, Ford et al. (1997) suggested that the cloned α_1a-AR subtype, which showed typical characteristics of the α_1a-AR in binding experiments, exhibited pharmacological characteristics as α_1L-AR in the functional experiments. In fact, several investigators have indicated that there is more similarity of pharmacological characteristics between α_1L- and α_1A-ARs than between α_1L- and α_1B-/α_1D-ARs (Williams et al., 1996; Daniels et al., 1996). In addition, α_1A-AR has recently been further subdivided into four isoforms based on their structural bases derived from alternative splicing of the single gene transcript (Hirasawa et al., 1995; Chang et al., 1998). Although the pharmacological profiles of these isoforms do not differ much, as so far reported (Hirasawa et al., 1995; Chang et al., 1998), one of the isoforms may possibly represent the α_1L-AR. In spite of this controversy, the existence of a distinct α₁-AR with low affinity for PZ has been widely accepted especially in functional studies (Bylund et al., 1994; Graham et al., 1996; Muramatsu et al., 1995; Kava et al., 1998). Therefore, we examined the affinity of KMD-3213 for native α₁-AR subtypes including α_1L-AR in functional experiments. KMD-3213 showed a prominent subtype selectivity maintaining high affinity for α_1A- and α_1L-ARs. The values of pA₂ were 10.0, 9.9, 7.7 and 8.3 for α_1A-, α_1L-, α_1B- and α_1D-ARs, respectively (Table 5). In rat caudal artery, KMD-3213 sometimes gave apparently insurmountable contractile responses even at 100 μM NS-49 especially at higher concentration of the antagonist (Figure 4a). However, we thought that this is due to its high affinity to the receptor in that tissue and that the antagonism by KMD-3213 is basically competitive in nature. This α_1A (and/or α_1L)-AR selectivity was not seen in both PZ and YM-617. Recently, however, several chemicals have been also developed as α_1A-AR selective antagonists, such as SNAP5089 (Lachnit et al., 1997), RS17053 (Ford et al., 1996), Rec15/2739 (Testa et al., 1996), and L762459 (O'Malley et al., 1998). These including KMD-3213 are targeted to medical therapeutics for diseases such as benign prostatic hypertrophy.

In conclusion, KMD-3213 is an α₁-AR antagonist that shows high affinity and selectivity for α_1A-AR and functional α_1L-AR subtypes compared with PZ or YM-617. Because of its high selectivity, KMD-3213 and its radioligand can be useful tools for analysing α₁-AR pharmacology in native tissues or recombinant receptors.

Abbreviations

AR

adrenoceptor

KMD-3213

(−)-(R)-1-(3-hydroxypropyl)-5-[2-[[2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl] amino]propyl]indoline-7-carboxamide

NA

noradrenaline

PZ

prazosin

YM-617

tamsulosin