Evaluation of α₁-adrenoceptors in the rabbit iris: pharmacological characterization and expression of mRNA

Abstract

1. Subtypes of α₁-adrenoceptor in rabbit iris have been examined in functional, binding and molecular biological experiments.

2. In functional studies, exogenous and endogenous noradrenaline produced contractions of the iris dilator muscle. The contractile responses to noradrenaline were competitively antagonized by a range of α₁-adrenoceptor antagonists (pA₂ values): prazosin (8.1), WB4101 (8.2), BMY7378 (5.9), YM617 (9.5), JTH-601 (8.8), HV723 (7.8) and KMD-3213 (9.8). The same order of inhibitory potency was seen in the adrenergic responses to electrical stimulation. This affinity profile corresponds well to that of the putative α_1L-adrenoceptor, which has been proposed in lower urinary tract tissues.

3. In binding studies on rabbit iris membrane however, prazosin, KMD-3213 and WB4101 displayed high affinity (pK_d or pK_i: 9.6, 10.3, 9.6, respectively), and BMY7378 displayed low affinity (pK_i: 6.9). These results show that the binding sites typically correspond to α_1A-adrenoceptor subtype in character, and we could not detect the significant amount of α_1L-adrenoceptor subtype.

4. The expression of the three distinct mRNAs that encode proteins of α_1a-, α_1b- and α_1d-adrenoceptors was studied using reverse transcription-polymerase chain reaction (RT–PCR). RT–PCR demonstrated the strongest expression of the α_1a-adrenoceptor, weak expression of the α_1b- adrenoceptor and undetectable expression of the α_1d-adrenoceptor.

5. The present study suggests that α_1A-adrenoceptor is a major subtype detectable in binding and RT–PCR studies in rabbit iris, but that the adrenergic contractions of iris dilator muscle are mediated via activation of α₁-adrenoceptor subtype having low affinity for prazosin and WB4101.

Introduction

Pharmacological and molecular cloning studies have established operational and structural heterogeneity among α₁-adrenoceptors (Minneman, 1988; Ford et al., 1994; Bylund et al., 1994). The current classification recognizes the existence of three α₁-adrenoceptors (α_1A, α_1B and α_1D) all of which meet well the criteria (i.e., sequence, second messenger and pharmacological profile: Hieble et al., 1995). A fourth α₁-adrenoceptor, α_1L-adrenoceptor, which is mainly based on the results of functional pharmacological studies, has been postulated (Holck et al., 1983; Flavahan & Vanhoutte, 1986; Muramatsu, 1992). However, the corresponding gene has not been identified yet. Being widely distributed in heart, liver, lower urinary tract tissues, vascular and ocular smooth muscle tissues, α₁-adrenoceptors play an important physiological role in peripheral organs (Lefkowitz & Caron, 1988; Minneman, 1988; Muramatsu, 1992; Ford et al., 1996; Miyamoto et al., 1997; Suzuki et al., 1997).

The iris, which controls the size of the pupil and thus regulates the amount of light admitted, is innervated by sympathetic and parasympathetic nerves; the former regulates the contraction of dilator muscle via α₁-adrenoceptors (Malmfors, 1965; Van Alphen et al., 1965; Narita & Watanabe, 1982; Konno & Takayanagi, 1986; McGregor, 1994) and the latter controls that of sphincter muscle via muscarinic receptors (Choppin et al., 1998) in many mammalian species. Thus, agonism of α₁-adrenoceptors in the iris dilator leads to mydriasis, conversely antagonism results in miosis. Ishikawa et al., (1996) reported that the α₁-adrenoceptor subtype in rabbit iris dilator has high affinity for prazosin, while that in the iris dilator of human or pigmented rabbit has low affinity for prazosin. Mittag & Tormay (1985) reported that the pK_d value of [³H]-prazosin is 9.3 in rabbit iris-ciliary body. However, because of a small amount of this tissue, very few binding studies have been made in the iris so far, and it still remains unclarified which subtype(s) of α₁-adrenoceptors are involved in the contraction of the dilator muscle.

The purpose of this study is to demonstrate the α₁-adrenoceptor subtype responsible for the rabbit iris dilator contraction in the functional study, and to characterize the α₁-adrenoceptor subtype of whole rabbit iris in the binding study and RT–PCR assay.

Methods

Animals

Tissues were obtained from adult male albino rabbits that weighed 2–2.5 kg. Rabbits were anaesthetized with intravenous sodium pentobarbital (50 mg kg⁻¹) and killed. The eyes were enucleated promptly and dissected under a surgical microscope to isolate iris in ice-cold modified Krebs-Henseleit solution of the following composition (mM): NaCl 112, KCl 5.9, MgCl₂ 1.2, CaCl₂ 2, NaHCO₃ 25, NaH₂PO₄ 1.2, and glucose 11.5.

Functional experiments

A strip of iris dilator muscle was prepared in approximately 2 mm width and 4 mm length and was mounted vertically in an organ bath containing 20 ml of modified Krebs-Henseleit solution, continuously bubbled with 95% O₂ and 5% CO₂ at 37°C. All preparations were equilibrated for 90 min with a resting tension of 50 mg before the experiments were begun. Changes in isometric tension were recorded on a pen-writing oscillograph (FJG-4128, Nihon Koden, Tokyo, Japan) in the following experiments.

Concentration-response curves for agonists were obtained by adding the drugs directly to the bath in cumulative fashion. The concentration-response curves for noradrenaline were obtained four times in the same strip at 60 min washout intervals. The second concentration-response curves were used as the control and α₁-adrenoceptor antagonists were added for 30 min before and during the third and fourth responses. In all experiments desipramine (0.1 μM), deoxycorticosterone acetate (1 μM) and propranolol (1 μM) were added in the bath to block neural and extraneural uptake of noradrenaline and to block β-adrenoceptors, respectively.

For the transmural electrical stimulation (TES), the preparation was placed between a pair of platinum electrodes. Gaps between the electrodes were wide enough to accommodate the strip and yet sufficiently narrow to stimulate the intramural nerve terminals effectively. To block presynaptic α₂-adrenoceptor, DG-5128 (10 μM) was added to the bath (Muramatsu et al., 1983). Although Yamanaka et al. (1984) showed that IC₅₀ value of this compound at α₁-adrenoceptors of rat brain homogenates is approximately 4 μM, we confirmed that noradrenaline-induced contractions of the rabbit dilator muscle are not affected by DG-5128 up to 10 μM in preliminary examination (data not shown). A train of rectangular pulses (7.5 mV intensity, 130 μs duration, 10 Hz frequency, for 10 s) was applied every 15 min. Antagonists to be tested were added to the organ chamber 30 min before the stimulation.

The affinities for α₁-adrenoceptor antagonists were estimated according to Arunlakshana & Schild (1959). Data were plotted as the −log [antagonist concentration] vs the log [concentration ratio (CR) −1], and pA₂ values with mean slopes and 95% confidence limits (95% CL) were calculated and first regression lines were drawn by least square method.

Binding experiments

For membrane preparation the irides were minced with scissors and homogenized in 20 volume of buffer (Tris HCl 50 mM, NaCl 100 mM, EDTA 2 mM, pH 7.4) with a polytron homogenizer (setting 8, 15 s×3) and centrifuged at 3000×g for 15 min. The supernatant was filtered through four layers of surgical gauze and subjected to centrifugation at 80,000×g for 30 min. The pellet was resuspended in ice-cold assay buffer (Tris HCl 50 mM, EDTA 1 mM, pH 7.4) and the resuspension was centrifuged at 80,000×g for 30 min again. All centrifugation was done at 4°C. The resultant pellet was resuspended in assay buffer as a membrane fraction and was immediately used for binding assay.

In saturation binding experiments, the membranes were incubated with various concentrations of [³H]-ligand ([³H]-prazosin; 30 pM–5 nM or [³H]-KMD; 10 pM–1 nM) for 45 min at 30°C with incubation volume 1 and 2 ml, respectively. Nonspecific binding was defined as binding in the presence of a masking ligand (0.3 μM YM617 for [³H]-prazosin and 0.3 μM prazosin for [³H]-KMD). In competition binding experiments, the membranes were incubated with about 100 pM [³H]-KMD and unlabelled drugs for 45 min at 30°C. Reactions were terminated by rapid filtration through Whatman GF/C filters presoaked in 0.3% polyethylenimine for 45 min. The filters were then washed four times with 4 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4) and dried. The filter-bound radioactivity was determined by liquid scintillation counting. All assays were performed 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). Displacement binding data were analysed by GraphPAD Prism. When the slope factor was close to unity, concentrations of competing agent producing 50% displacement of [³H]-KMD (IC₅₀) were converted to K_i values with the Cheng & Prusoff approximation (1973). Protein determination was assayed according to the method of Bradford (1976) with bovine serum albumin as standard.

RT–PCR assays

The iris was frozen in liquid nitrogen immediately after isolation and then stored at −80°C until extraction of RNA. Total cellular RNA was extracted according to the procedure of Chomczynski & Sacchi (1987). All RNA transcripts for three adrenoceptor subtypes were synthesized from the appropriate plasmid DNA with the use of T7 RNA polymerase (Gibco BRL). To produce the cDNA, total RNA (500 ng) from iris and cRNA (300 pg) from clones were individually reverse transcribed by moloney murine leukaemia virus reverse transcriptase (Gibco BRL) with random hexamer. Three μl of cDNA was amplified in a 25 μl PCR mixture containing (mM) KCl 50, Tris-HCl 10, pH 8.3, MgCl₂ 2, 200 μM each of dNTP, 0.25 μM sense and antisense primers and 2.5 U Pwo DNA polymerase (Boehringer). The subtype specific primers were designed from published sequences of rabbit α_1a- and α_1d-adrenoceptors (Miyamoto et al., 1997; Suzuki et al., 1997) and unpublished sequence of rabbit α_1b-adrenoceptor, as follows: α_1a: 5′-CATCGTGGTCGGCTGCTTCGTC-3′, 5′-GGCTGTAGTGCAGGCTGATT-3′, α_1b: 5′-AGGAGCCGGCACCCAATGATGA-3′, 5′-GGCACTGGCACCCGAGGAT-3′, α_1d: 5′-CTCCGTGCGCCTGCTCAAGT-3′, 5′-GGGTAGATGAGTGGGTTCAC-3′. PCR was performed in a Perkin-Elmer DNA Thermal Cycler 2400 with the following cycle parameters: one denaturation cycle for 3 min at 98°C; 40 cycles of 30 s at 95°C, annealing for 10 s and extension for 15 s at 72°C. Annealing temperatures were 68°C for the α_1a-adrenoceptor, 64°C for the α_1b-and α_1d-adrenoceptor. For each experiment, negative controls were amplified, in which all of the components except reverse transcriptase were included. The PCR products were separated on 3.5% polyacrylamide gels and stained with ethidium bromide.

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) and tamsulosin hydrochloride (YM-617) from Kissei Pharmaceutical Co. Ltd. (Matsumoto, Japan); 3-[N-[2-[4-hydroxy-2-isopropyl-5-methylphenoxy] ethyl] -N-methylaminomethyl] - 4-methoxy-2,5,6-trimethylphenol hemifumarate (JTH-601) from Japan Tobacco Inc. (Osaka, Japan); α-ethyl-3,4,5-trimethoxy-α-[3-[ [2-2-methoxyphenoxy]ethyl] -amino] -propyl] benzeneacetonitrile fumarate] (HV723) from Hokuriku Seiyaku (Katsuyama, Japan); [2-[2-[4,5-dihydro-1H-imidazol-2-yl]1-phenylethyl] pyridine dihydrochloride sesquihydrate] (DG-5128) from Daiichi Seiyaku (Tokyo, Japan); prazosin hydrochloride, (−)-noradrenaline bitartrate, (−)-phenylephrine hydrochloride, oxymetazoline and desipramine hydrochloride, from Sigma (St. Louis, U.S.A.); WB4101 and BMY7378 from Research Biochemicals Inc. (Natick, U.S.A.); (−)-propranolol hydrochloride and deoxycorticosterone acetate from Nakalai (Kyoto, Japan); clonidine hydrochloride from Funakoshi (Tokyo, Japan); [³H]-KMD-3213 (49 Ci mmol⁻¹), from Amersham (England); and [³H]-prazosin (77.2 Ci mmol⁻¹) from NEN (Boston, U.S.A.).

Results

Functional study

Figure 1 shows the concentration-response curves for noradrenaline, phenylephrine, oxymetazoline and clonidine in the rabbit iris dilator muscle. All agonists produced concentration-dependent contraction in dilator muscle. The maximal amplitude of contraction induced by phenylephrine was almost equal to that induced by noradrenaline, whereas those by oxymetazoline or clonidine were smaller than that induced by noradrenaline. The pEC₅₀ values and relative contractions of these agonists are summarized in Table 1, in which the maximal amplitude of contraction was compared with that induced by noradrenaline in the same strip. The following order of potency was observed: oxymetazoline > noradrenaline > phenylephrine > clonidine. Several α₁-adrenoceptor antagonists (prazosin, WB4101, BMY7378, YM617, JTH-601, HV723 and KMD-3213) were tested for their ability to inhibit noradrenaline-induced responses and their functional affinity estimates (pA₂ values) are summarized in Table 2. The concentration-response curves for noradrenaline were antagonized by these compounds in a concentration-dependent manner, with parallel rightward displacements. Figure 2 shows representative results with prazosin and KMD-3213. The Schild slopes were not significantly different from unity for all these antagonists, indicating that the antagonism was competitive in nature. KMD-3213, YM617 and JTH-601 displayed higher affinity than the other antagonists (Table 2). Rauwolscine (1 μM) had no effect on noradrenaline-induced contraction (data not shown).

Figure 1.

Concentration-response curves for four α-adrenoceptor agonists in isolated rabbit iris dilator muscle. The maximal contraction induced by 100 μM noradrenaline (267.2±19.4 mN; n=8) was taken as 100% in each preparation. The data shown are means±s.e.mean of 4–8 experiments. For pEC₅₀ values and maximal contractions see Table 1.

Table 1.

Contractile parameters for various α-adrenoceptor agonists in isolated rabbit iris dilator

Table 2.

Functional affinities for α₁-adrenoceptor antagonists in isolated rabbit iris dilator

Figure 2.

Concentration-response curves of isolated rabbit iris dilator muscle for noradrenaline and effects of prazosin (a) and KMD-3213 (b). Responses were plotted as percentages of maximal contractile response for noradrenaline in control. The data shown are means±s.e.mean of 3–6 experiments. For pA₂ values see Table 2.

TES at 10 Hz for 10 s produced a monophasic and transient contraction of dilator muscle. The responses to TES were abolished by 0.5 μM tetrodotoxin. The α₁-adrenoceptor antagonists mentioned above inhibited but not abolished the contraction induced by TES; thus 15% of contractile amplitude remained as the resistant component (Muramatsu et al., 1994). Their functional affinities (pIC₅₀ which was estimated at the level of 50% reduction of α₁-adrenoceptor antagonist-sensitive contraction) are also summarized in Table 2. The rank order of pIC₅₀ was the same as pA₂ values estimated in noradrenaline-response.

Binding study

Figure 3 shows representative data obtained in saturation experiments with [³H]-prazosin (a) and [³H]-KMD (b), respectively. Both [³H]-prazosin and [³H]-KMD meet the criteria for binding to a single set of sites. The binding isotherms for these ligands were hyperbolic, as shown in Figure 3. The pK_d values and maximum binding sites (B_max) of both ligands are shown in Table 3. Although we could not carry out many experiments because of limitation of tissue amount, there was no difference between the two independent experiments (Experiment 1 and Experiment 2). The pK_d values of [³H]-prazosin and [³H]-KMD are 9.6 and 10.3 (mean of two experiments), respectively, and B_max values of [³H]-prazosin and [³H]-KMD were almost same (Table 3). The specific bindings of [³H]-KMD were displaced by simultaneous addition of nonradioactive ligands (Figure 4). The slope factors of the drugs were not significantly different from unity, suggesting the presence of a single type of binding site. The pK_i values for drugs in two independent experiments were almost same (Table 3).

Figure 3.

Saturation experiments of [³H]-prazosin (a) and [³H]-KMD (b) binding to rabbit iris membranes. The data are obtained from a single experiment (Experiment 1) where each point is the mean of duplicate determinations. Another experiment was also carried out with different membrane preparation and the data obtained from two independent experiments were shown in Table 3.

Table 3.

Binding parameters estimated with [³H]-prazosin and [³H]-KMD in rabbit iris membranes

Figure 4.

Displacement of [³H]-KMD binding to rabbit iris membranes by α₁-adrenoceptor antagonists. The concentration of [³H]-KMD used was 100 pM. The data are obtained from a single experiment (Experiment 2) where each point is the mean of duplicate determinations. The data obtained from two independent experiments were shown in Table 3.

RT–PCR study

Figure 5 illustrates the results of a representative experiment using total RNA from rabbit iris and cRNAs from cloned α₁-adrenoceptor subtypes. The RT–PCR products derived from clone cRNAs are observed for α_1a-adrenoceptor at 442 bp (A, lane 4), α_1b-adrenoceptor at 549 bp (B, lane 4) and α_1d-adrenoceptor at 210 bp (C, lane 4), respectively. The cDNA generated from total RNA of irides was amplified and expression of α₁-adrenoceptor subtypes mRNA in rabbit iris is shown in lanes 2. The α_1a-adrenoceptor specific oligonucleotide primers amplified a large amount of cDNA (A, lane 2). A faint band was generated with the α_1b-adrenoceptor specific primers (B, lane 2) while no detectable PCR product was amplified with the α_1d-adrenoceptor specific primers (C, lane 2). All PCR fragments corresponded to the predicted fragment size for each receptor subtype. No PCR product was seen in the control experiments which did not contain reverse transcriptase in the reverse transcriptase-reaction (A,B,C, lanes 3 and 5), suggesting no DNA contamination was present. Two other experiments conducted under same conditions showed the identical results.

Figure 5.

Polyacrylamide gel electrophoresis of 5 μl of RT–PCR product. Lane 1 shows DNA size markers ΦX174/HaeIII. Lanes 2 and 3 represent adrenoceptor RT–PCR products from iris total RNA. Lanes 4 and 5 represent α_1a-, α_1b-, and α_1d-adrenoceptor RT–PCR products from 300 pg of each adrenoceptor clone cRNA. Bands of lane 4 442 bp (A), 549 bp (B), 210 bp (C) is a product of the α_1a-, α_1b- and α_1d-adrenoceptor clone cRNA respectively. No PCR products were seen in the control experiments, which contained no reverse transcriptase in the reverse transcriptase-reaction (lanes 3 and 5).

Discussion

In the functional study, a series of α-adrenoceptor agonists and antagonists, which should be able to discriminate the subtype involved (Hieble et al., 1995; Muramatsu et al., 1998), were used to characterize the α-adrenoceptors in the dilator smooth muscle of rabbit iris. Noradrenaline and phenylephrine acted as full agonists, while oxymetazoline and clonidine behaved as partial agonists (Figure 1 and Table 1). The contractile responses to noradrenaline and TES were inhibited in a concentration-dependent and competitive fashion by several α₁-adrenoceptor selective antagonists (Figure 2). Whereas the responses to noradrenaline were not inhibited by 1 μM of rauwolscine (a selective antagonist for α₂-adrenoceptor). These results indicate that the adrenergic responses in rabbit iris dilator are mediated through α₁-adrenoceptors. However, the potencies or affinities varied among the agonists or antagonists used (Tables 1 and 2), suggesting an involvement of unique α₁-adrenoceptors in the adrenergic responses. Since Schild slopes for tested antagonists were close to unity and the range of effective concentrations of tested agonists were less than three log units, it appears that the adrenergic contractions are predominantly mediated by a single α₁-adrenoceptor subtype. In the series of antagonists tested, KMD-3213 (pA₂: 9.8), YM617 (9.5) and JTH-601 (8.8) showed high affinity, while WB4101 (8.2), prazosin (8.1), HV723 (7.8) and BMY7378 (5.9) showed low affinity. These affinity estimates of antagonists correlated most closely with those in lower urinary tract tissues of rabbit or human (pA₂: prazosin 7.85–8.26; WB4101 8.4–8.9; BMY7378 6.4; YM617 9.31–9.49; JTH-601 8.8; HV723 7.63–8.05; KMD-3213 9.9–10.05) (Honda et al., 1985; Ford et al., 1996; Yamagishi et al., 1996; Leonardi et al., 1997; Takahashi et al., 1998; Murata et al., 1999), where putative α_1L-adrenoceptor has been proposed in the adrenergic responses. Furthermore, the affinity values for these antagonists against the responses to TES showed the same trend (Table 2). These results suggest that exogenous and endogenous noradrenaline acts on the same subtype (presumably α_1L-adrenoceptor) to cause contraction in rabbit iris dilator.

The α_1L-adrenoceptor subtype is characterized according to low affinity for prazosin and other antagonists (Flavahan & Vanhoutte, 1986; Muramatsu et al., 1990). Marshall et al. (1996) argued that one of the main problems in determining whether prazosin does distinguish between α₁-adrenoceptor subtypes is that the pA₂ values published cover a continuous range of values rather than falling into discrete groups. For example, pA₂ values of 8.3, 8.2 and 8.1 have been documented for α₁-adrenoceptors in the rabbit trigone, urethra and prostate, respectively (Honda et al., 1985), 8.5 in rabbit prostate (Williams & Clarke, 1993), 8.9 in rat anococcygeus (Ford et al., 1993) and rat portal vein (Schwietert et al., 1991), 9.2 in rat epididymal vas deferens, spleen and portal vein (Burt et al., 1995), 9.3, 9.5, and 9.6 in rat perfused kidney, perfused mesentery and aortic rings respectively (Ford et al., 1996) and 9.8 in rat aorta (Kenny et al., 1995). In this study, however, the pA₂ value of 8.1 obtained for prazosin on the rabbit iris dilator is low enough to show the involvement of α_1L-adrenoceptors in the contractile response of iris dilator muscle.

In the binding study, however, not only [³H]-KMD but also [³H]-prazosin bound to the iris membranes with subnanomolar high affinity (pK_d: 10.3 for [³H]-KMD and 9.6 for [³H]-prazosin, respectively) and with the same density (Figure 3 and Table 3). Furthermore, [³H]-KMD binding sites were recognized as prazosin- and WB4101- high affinity sites (Figure 4 and Table 3). Originally, KMD-3213 was reported as an α_1A-adrenoceptor-selective antagonist (Shibata et al., 1995). In the experiments with human recombinant adrenoceptors, KMD-3213 showed a pK_i value of 10.4 for the α_1a-adrenoceptor, but has 580- and 56 fold lower affinity for α_1b and α_1d-adrenoceptors, respectively. Recently, it was also reported that KMD-3213 shows high affinity not only for α_1a- but also for α_1L-adrenoceptor (pA₂: 9.9) (Murata et al., 1999). This high selectivity of KMD-3213 to α_1L-adrenoceptor was confirmed in the present functional studies (Table 2). So it is likely that Bmax of [³H]-KMD consist of major α_1A and minor α_1L site, whereas considering the result from RT–PCR studies, B_max of [³H]-prazosin consist of major α_1A and minor α_1B site. Although we could not carry out further analysis because of limitation of tissue amount, the results obtained clearly show that the binding sites correspond mainly to α_1A-adrenoceptor subtype in character, and that the significant amount of α_1L-adrenoceptor subtype is undetectable.

The results on mRNA expression of α₁-adrenoceptor subtypes agrees with the result of binding experiments. RT–PCR studies demonstrated that expression of mRNA for the α_1a-adrenoceptor is major and that those of the α_1b- and α_1d-adrenoceptors are minor or negligible, respectively. Although mRNA levels do not always correlate with protein expression, the predominance of mRNA for α_1a-adrenoceptor suggests that this receptor may play an important role in irideal function.

Thus, there is a discrepancy for α₁-adrenoceptor subtypes between functional and binding or RT–PCR studies. How do we consider this matter? First of all, we must consider experimental conditions. Williams et al. (1996) demonstrated that in binding studies of the human cloned α_1A-adrenoceptor, temperature and cellular integrity appear to influence affinity estimates for several key antagonists, resulting in an apparent profile as α_1L-adrenoceptor. We tested various conditions including those employed in this study, however, rabbit cloned α_1A-adrenoceptor expressed in Chinese hamster ovary (CHO) cells displayed the binding affinity profile as α_1A-adrenoceptor (data not shown).

Second, characteristics of compounds such as high lipophilicity, chemical instability and extra binding sites, are likely to underestimate the affinity constant. For example, a low affinity estimate of S-niguldipine in the human lower urinary tract is probably influenced by its high lipophilicity (Ford et al., 1996). For another example, the human α₁-acid glycoprotein can inhibit prazosin and WB4101 binding to α_1A-adrenoceptors and therefore reduce their affinity for these receptors (Chiang et al., 1991; Qin & Øie, 1994). However, because the Schild regressions performed for all antagonists used in our experiments validated that the inhibition curve design and the slopes were close to unity, these influences may be negligible.

Third, in most binding studies, the concentrations of [³H]-prazosin were up to 1 nM and then a single high affinity constant for prazosin (pK_d close to 10) has been estimated. However, it should be noted that affinity constant (K_B) of the α_1L-adrenoceptors found in the functional studies is close to 10 nM. Thus, it is likely that α_1L-adrenoceptors will be overlooked when low concentrations (<1 nM) of the [³H]-prazosin are used. Although in this study we used up to 5 nM of [³H]-prazosin, revealing the apparently saturated binding to α₁-adrenoceptors of rabbit iris (Figure 3a), this concentration might not be enough to detect α_1L-adrenoceptor subtype.

Fourth, it is likely that α_1A-adrenoceptor is a predominant subtype while α_1L-adrenoceptor is a minor in whole rabbit iris. The iris consists of four layers; anterior border layer, stroma and sphincter muscle layer, anterior epithelium and dilator muscle layer, posterior pigmented epithelium layer. In these, the dilator muscle is a very minor element (in human; 60 μm long and 7 μm wide) whereas the major structural element is the stroma which contains the vessels, nerves and cellular compartments (Bron et al., 1997). Previous reports showed that prazosin and bunazosin lower intraocular pressure in rabbits by a reduction in the rate of aqueous humor formation, which could occur by a vascular mechanism without affecting the pupillary diameter (Krupin et al., 1980; Green & Hatchett, 1987; Nishimura et al., 1993; Hong et al., 1998). These results suggest that there are at least two sets of α₁-adrenoceptors; one is high sensitive to prazosin in blood vessels and another shows low affinity to prazosin located in iris dilator muscle. Thus, it may be possible to speculate that α_1A-adrenoceptor distributes in the stroma, probably in the vessels, and shares the major part of α₁-adrenoceptor in whole iris while α_1L-adrenoceptor locates in the dilator muscle, as suggested in this study, and shares the small part.

Finally, a possible explanation for the discrepancy between functional and binding or molecular experiments is based on findings that functional estimates of antagonist affinity for the α_1a-adrenoceptor correlate closely with those established for the α_1L-adrenoceptor (Ford et al., 1997). Ford et al. compared binding affinities estimated by displacement of [³H]-prazosin in membrane homogenates of CHO cells stably expressing the human α_1a-, α_1b-, and α_1d-adrenoceptors with affinity estimates obtained functionally in identical cells by measuring inhibition of noradrenaline-stimulated accumulation of [³H]-inositol phosphates. And they suggested that upon functional pharmacological analysis, the cloned α_1a-adrenoceptor displays pharmacological recognition properties consistent with those of the putative α_1L-adrenoceptor. Recently, four isoforms of the α_1a-adrenoceptor generated by alternative splicing, which differ in length and sequence at the carboxy-terminal region, have been identified in human (Hirasawa et al., 1995; Chang et al., 1998). The α_1a-adrenoceptor of rabbit also have at least three splice variants (unpublished result), however, the physiological significance of α_1a-adrenoceptor splice variants is presently unknown. Since the isoforms differ only in their carboxy-terminal regions, they may differ in their preference for G-protein coupling or in rates of desensitization mediated by phosphorylation. Recent data have implicated that some splice variants may demonstrate α_1L-adrenoceptor pharmacology in functional studies (Chang et al., 1998). However, why functional profile differs from that obtained in binding study requires further explanation.

In conclusion, we have found that α_1A-adrenoceptor is a major subtype in the α₁-adrenoceptors distributing in rabbit iris from binding study and that mRNA encoding this subtype is predominant in whole rabbit iris. While the dilator muscle, one of the constituents of the iris contracts via activation of α₁-adrenoceptor having low affinity for prazosin, WB4101 and BMY7378. This discrepancy is highly debatable, including the controversy whether or not the gene encoding α_1L-adrenoceptor exist. Also in human, α₁-adrenoceptor of iris dilator is characterized as α_1L-adrenoceptor (pA₂: prazosin 7.3) (Ishikawa et al., 1996). Therefore, rabbit may be the good strain for the drug research which is relevant to human ocular therapeutics. For example, α₁-adrenoceptor antagonists which have high affinity to the iris dilator may be available to reverse the effect of mydriasis rapidly and safely after diagnostic mydriasis (Mapstone, 1977; Relf et al., 1988; Allinson et al., 1990). Further study may reveal subtypes of α₁-adrenoceptor in ocular tissues with different locations and profiles for various agonists and antagonists, leading to development of a subtype selective drugs with therapeutic advantages.

Abbreviations

CHO

Chinese hamster ovary

RT–PCR

reverse transcription-polymerase chain reaction

TES

transmural electrical stimulation