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Research ArticleArticle

Murine Alpha 1-Adrenoceptor Subtypes. I. Radioligand Binding Studies

Ming Yang, Jens Reese, Susanna Cotecchia and Martin C. Michel
Journal of Pharmacology and Experimental Therapeutics August 1998, 286 (2) 841-847;

Abstract

Alpha1-adrenoceptors were identified in murine tissues by [3H]prazosin saturation binding studies, with a rank order of cerebral cortex > cerebellum > liver > lung > kidney > heart > spleen, with the spleen not exhibiting detectable expression. Competition binding studies were performed with 5-methylurapidil, BMY 7378, methoxamine, (+)-niguldipine, noradrenaline, SB 216469 and tamsulosin. On the basis of monophasic low-affinity competition by BMY 7378,alpha1D-adrenoceptors were not detected at the protein level in any tissue. On the basis of competition studies with thealpha1A/alpha1B-discriminating drugs, alpha1B-adrenoceptors appeared to be the predominant or even the sole subtype in murine liver, lung and cerebellum, whereas murine cerebral cortex and kidney contained ∼30% and 50% of alpha1A-adrenoceptors, respectively. The affinities of the various competitors in the murine tissues were quite similar to those reported from other species. The ratio of high- and low-affinity sites for tamsulosin did not in all cases match the percentages of alpha1A- andalpha1B-adrenoceptors detected by the other competitors; however, the low-affinity component of the tamsulosin competition curves was abolished in the cerebral cortex ofalpha1B-adrenoceptor knockout mice. Treatment with chloroethylclonidine (10 μM, 30 min, 37°C) inactivated the alpha1-adrenoceptors in all tissues by >75%. When the concentration-dependent inactivation of tissue alpha1B-adrenoceptors (liver) and tissue alpha1A-adrenoceptors (cerebral cortex from alpha1B-adrenoceptor knockout mice) was compared, alpha1A-adrenoceptors were only slightly less sensitive toward chloroethylclonidine thanalpha1B-adrenoceptors. We conclude that murine tissues express alpha1A- andalpha1B-adrenoceptors, which are largely similar to those in other species. However, the tissue-specific distribution of subtypes may differ from that of other species.

Alpha1-adrenoceptors mediate many of the physiological functions of the sympathoadrenal transmitters adrenaline and noradrenaline (Ruffolo and Hieble, 1994). Therefore, they are important drug targets, e.g., in the cardiovascular and urogenital systems (Ruffolo et al., 1995). In recent years it has become clear that at least three subtypes of alpha1-adrenoceptors exist, which are designated alpha1A,alpha1B andalpha1D (Hieble et al., 1995;Michel et al., 1995). The genes and/or cDNAs encoding these three subtypes have been cloned in rats and humans (Hieble et al., 1995), and species homologs for some subtypes have additionally been cloned in hamsters, cows, rabbits and mice (Cotecchiaet al., 1988; Schwinn et al., 1990;Alonso-Llamazares et al., 1995; Miyamoto et al., 1997; Suzuki et al., 1997). The pharmacological characterization of tissuealpha1-adrenoceptor subtypes has mainly been performed in rats (Michel et al., 1995). Some data on the characterization of tissuealpha1-adrenoceptor subtypes are available in humans (Gross et al., 1989; Lepor et al., 1993; Michel et al., 1996), guinea pigs (Garcia-Sainzet al., 1992; Haynes and Pennefather, 1993, Büscheret al., 1996) and cows (Büscher et al., 1996) but only very few in mice (Garcia-Sainz et al., 1994).

Transgenic techniques are a powerful tool of molecular pharmacology because they allow the study of phenotypes that are caused by well-defined genotypes (Chien, 1996). The most frequently used species in the generation of transgenic animals is the mouse. In thealpha1-adrenoceptor field, transgenic mice have been generated that overexpress constitutively activealpha1B-adrenoceptors in their myocardium (Milano et al., 1994). More recently, knockout mice have been generated that lack endogenousalpha1B-adrenoceptors (Cavalli et al., 1997). Studies of these mice have yielded intriguing observations, but their interpretations are limited by the scarcity of knowledge regarding the physiological characteristics of murine alpha1-adrenoceptors relative to those of other species. Therefore, we have characterizedalpha1-adrenoceptors in a variety of murine tissues by using competition binding studies with several subtype-selective drugs and receptor inactivation studies with chloroethylclonidine.

Materials and Methods

Tissue preparation.

Mice of either sex (strain HLG, 25–35 g) were obtained from the central animal breeding facility at the University of Essen. In some experimentsalpha1B-adrenoceptor knockout mice (C57 × 129 hybrids) were used, which have been described in detail previously (Cavalli et al., 1997). Mice were sacrificed by decapitation, and the cerebral cortex, cerebellum, heart, kidney, liver, lung and spleen were removed rapidly. The tissues were macroscopically cleared from adhering connective tissue, rapidly frozen in liquid nitrogen and stored at −80°C for up to 3 months until analysis.

Radioligand binding.

Radioligand binding with the use of [3H]prazosin as the ligand was performed as previously described (Michel et al., 1993a). Briefly, aliquots of the membrane suspensions were incubated in a total volume of 1000 μl of binding buffer (50 mM tris [hydroxymethyl]aminomethane, 0.5 mM ethylenediamine tetraacetate at pH 7.5) for 45 min at 25°C. In competition binding experiments with agonists, 100 μM guanosine triphosphate was always added to prevent guanosine diphosphate-dependent formation of agonist high-affinity states. The incubation was terminated by rapid vacuum filtration over Whatman GF/C filters, and each filter was washed twice with 10 ml of binding buffer. After drying of the filters for 1 h at 60°C, 4 ml of scintillator (Quickszint 1, Zinsser, Frankfurt, Germany) was added to each filter, and after vigorous shaking of each sample, the radioactivity on the filters was quantified in a scintillation counter at 42% efficiency. Nonspecific binding was defined as binding in the presence of 10 μM phentolamine. In some experiments, membrane preparations were treated with the indicated concentrations of chloroethylclonidine or vehicle for 30 min at 37°C, followed by two washout centrifugations before incubation with the radioligand.

Chemicals.

Dye reagent for the protein assay was purchased from Bio-Rad (Munich, Germany); methoxamine HCl and (-)-noradrenaline bitartrate from Sigma (Deisenhofen, Germany); chloroethylclonidine HCl, 5-methylurapidil, (+)-niguldipine HCl and BMY 7378 from Research Biochemicals Inc. (Natick, MA); and [3H]prazosin (specific activity, 80 Ci/mmol) from New England Nuclear (Dreieich, Germany). The following drugs were gifts of the respective companies: tamsulosin HCl [(-)-isomer, formerly known as YM 617; Yamanouchi Pharmaceutical Co., Tokyo, Japan], SB 216469 (formerly known as Rec 15/2739; Recordati, Milan, Italy) and phentolamine HCl (Ciba-Geigy, Basel, Switzerland).

Data analysis.

Data are shown as means ± S.E.M. ofn experiments. Saturation binding experiments were analyzed by fitting rectangular hyperbolic functions to the experimental data. Competition binding experiments were analyzed by fitting monophasic and biphasic sigmoidal functions to the experimental data; a two-site fit was accepted only when it resulted in a significant improvement over the one-site fit as assessed by an F test. Kivalues were calculated from the IC50 values in the binding and functional experiments according to the equationKi = IC50/[(L/Kd)], whereL is the concentration of radioligand andKd is its affinity. All curve-fitting procedures were performed by using the InPlot program (GraphPAD Software, San Diego, CA). Statistical significance of differences was assessed by two-tailed t tests by using the InStat program (GraphPAD Software), and a P < .05 was considered significant.

Results

[3H]Prazosin identified specific, saturable, high-affinity binding sites in the murine cerebral cortex, cerebellum, liver, lung, kidney and heart (table1), but no quantifiable specific binding was detected in murine spleen (data not shown).Kd values for [3H]prazosin were similar in all tissues and amounted to 54 ± 14 pM in cerebral cortex, 87 ± 15 pM in cerebellum, 78 ± 11 pM in liver, 67 ± 10 pM in lung, 73 ± 11 pM in kidney and 66 ± 12 pM in heart (n = 4–5 each).

View this table:
Table 1

α1-Adrenoceptor density in control and chloroethylclonidine-treated murine tissues

The murine alpha1-adrenoceptors were characterized pharmacologically by competition binding experiments by using the subtype-selective agonists noradrenaline and methoxamine and the antagonists 5-methylurapidil, BMY 7378, (+)-niguldipine, SB 216469 and tamsulosin. In murine liver, all agonists and antagonists competed for [3H]prazosin binding with steep and monophasic curves of low affinity (table2, figs.1-7), indicating the presence of a homogeneous population ofalpha1B-adrenoceptors.

View this table:
Table 2

Competition binding parameters in murine liver

In murine cerebral cortex, competition curves for thealpha1D-selective BMY 7378 were steep, monophasic and of low affinity (table 3, fig. 2). Competition curves for 5-methylurapidil, methoxamine, (+)-niguldipine and tamsulosin were significantly better explained by a two-site model than a one-site model in most cases; the percentage of high-affinity sites for these compounds ranged between 14% and 43% (table 3, figs. 1,3, 4 and7). Although competition curves for noradrenaline and SB 216469 also were slightly shallow, they were not significantly better explained by a two-site than a one-site model in at least half of all experiments; the calculated affinities of both compounds were low (table 3, Figs.5 and 6). In cerebral cortex of alpha1B-adrenoceptor knockout mice, the competition curves for (+)-niguldipine and tamsulosin were considerably steeper than in control mice; they were no longer significantly better explained by a two-site model in most cases, and the calculated affinity of both compounds was high [−logKi (+)-niguldipine, 9.40 ± 0.18 and tamsulosin, 10.29 ± 0.27; n = 4 each; fig. 8).

View this table:
Table 3

Competition binding parameters in murine cerebral cortex

Figure 2
Figure 2

BMY 7378 competition for [3H]prazosin binding to murine cerebral cortex (open squares), kidney (filled squares), lung (open circles) and liver (filled circles). Data are means of the total number of experiments indicated in tables 2, 3, 5and 6, which also contain the quantitative analysis of the data.

Figure 1
Figure 1

5-Methylurapidil competition for [3H]prazosin binding to murine cerebral cortex (open squares), kidney (filled squares), lung (open circles) and liver (filled circles). Data are means of the total number of experiments indicated in tables 2, 3, 5 and 6, which also contain the quantitative analysis of the data.  

Figure 3
Figure 3

Methoxamine competition for [3H]prazosin binding to murine cerebral cortex (open squares), cerebellum (filled triangles), kidney (filled squares), lung (open circles) and liver (filled circles). Data are means of the total number of experiments indicated in tables 2 through 6, which also contain the quantitative analysis of the data.  

Figure 4
Figure 4

(+)-Niguldipine competition for [3H]prazosin binding to murine cerebral cortex (open squares), cerebellum (filled triangles), kidney (filled squares), lung (open circles) and liver (filled circles). Data are means of the total number of experiments indicated in tables 2 through 6, which also contain the quantitative analysis of the data.

Figure 7
Figure 7

Tamsulosin competition for [3H]prazosin binding to murine cerebral cortex (open squares), cerebellum (filled triangles), kidney (filled squares), lung (open circles) and liver (filled circles). Data are means of the total number of experiments indicated in tables 2 through 6, which also contain the quantitative analysis of the data.

Figure 5
Figure 5

Noradrenaline competition for [3H]prazosin binding to murine cerebral cortex (open squares), cerebellum (filled triangles), kidney (filled squares), lung (open circles) and liver (filled circles). Data are means of the total number of experiments indicated in tables 2 through 6, which also contain the quantitative analysis of the data.  

Figure 6
Figure 6

SB 216469 competition for [3H]prazosin binding to murine cerebral cortex (open squares), cerebellum (filled triangles), kidney (filled squares), lung (open circles) and liver (filled circles). Data are means of the total number of experiments indicated in tables 2 through 6, which also contain the quantitative analysis of the data.

Figure 8
Figure 8

(+)-Niguldipine (filled squares) and tamsulosin (open circles) competition for [3H]prazosin binding to murine cerebral cortex ofalpha1B-adrenoceptor knockout mice. Data are means ± S.E.M. of four experiments.

In murine cerebellum, competition curves for methoxamine, (+)-niguldipine, noradrenaline and SB 216469 were not significantly better explained by a two-site than a one-site model in at least half of all experiments, and the calculated affinities were low (table4, figs. 3-6). In contrast, the competition curves for tamsulosin were more shallow and significantly better explained by a two-site model in all cases (table 4, fig.7).

View this table:
Table 4

Competition binding parameters in murine cerebellum

In murine kidney, BMY 7378 and noradrenaline had steep and monophasic competition curves in almost all cases, and the calculated affinities were low (table 5, figs. 2 and 5). In contrast, 5-methylurapidil, methoxamine, (+)-niguldipine and SB 216469 had shallow and biphasic competition curves; the percentage of high-affinity sites for all compounds was similar and amounted to 48% to 55% (table 5, figs. 1, 3, 4 and 6). Although the competition curves for tamsulosin also were shallow, they were significantly better fitted to a two-site than a one-site model in only two of four experiments (table 5, fig. 7).

View this table:
Table 5

Competition binding parameters in murine kidney

In murine lung, BMY 7378, methoxamine, (+)-niguldipine, noradrenaline and SB 216469 had steep and monophasic competition curves in the majority of cases; the calculated affinities for all compounds was low (table 6, figs. 2-6). In contrast, 5-methylurapidil and tamsulosin had shallow and biphasic competition curves, which detected 54% to 59% high-affinity sites (table 6, figs.1 and 7).

View this table:
Table 6

Competition binding parameters in murine lung

Treatment with chloroethylclonidine (10 μM, 30 min, 37°C) significantly reduced the density of detectablealpha1-adrenoceptors in all tissues (table1). Thus, almost complete inactivation was observed in the liver and kidney, an ∼90% inactivation in the cerebral cortex and heart and an ∼75% to 80% inactivation in the cerebellum and lung. AlthoughKd values could not be reliably calculated in experiments with very extensive inactivation and/or small control receptor densities, no major change of apparent [3H]prazosin Kdvalues was seen in the remaining experiments (data not shown).

To resolve the discrepancy betweenalpha1B-adrenoceptor estimates in the competition binding studies and the chloroethylclonidine experiments, the concentration-dependent inactivation of murine tissuealpha1-adrenoceptor subtypes by chloroethylclonidine was investigated. For these experiments, liver tissue from control mice served as a source ofalpha1B-adrenoceptors, whereas cerebral cortex from alpha1B-adrenoceptor knockout mice served as a source ofalpha1A-adrenoceptors. Chloroethylclonidine (0.1–10 μM) concentration-dependently activatedalpha1-adrenoceptors in both preparations (fig. 9). Although the percentage of inactivation was significantly smaller foralpha1A- than foralpha1B-adrenoceptors at each tested concentration of chloroethylclonidine, the differences were only small, and 10 μM chloroethylclonidine inactivated ∼90% ofalpha1A-adrenoceptors.

Figure 9
Figure 9

Concentration-dependent inactivation of murinealpha1A- andalpha1B-adrenoceptors by chloroethylclonidine. Membrane preparations from murine liver from normal mice were used as a homogeneous source ofalpha1B-adrenoceptors (filled circles) and from cerebral cortex fromalpha1B-adrenoceptor knockout mice as a source of alpha1A-adrenoceptors (open squares). They were incubated in the absence (control) or presence of the indicated concentrations of chloroethylclonidine for 30 min at 37°C, followed by two washout centrifugations. Thereafter,alpha1-adrenoceptor density was determined by [3H]prazosin saturation binding experiments. Data are mean ± S.E.M. of four experiments.Alpha1-adrenoceptor density under control conditions in liver and cerebral cortex was 92 ± 17 and 59 ± 4 fmol/mg protein, respectively. * and **, P < .05 and .01, respectively, in a two-tailed t test compared with liver.

Discussion

The tissue- and cell type-specific distribution ofalpha1-adrenoceptor subtypes can be studied at the mRNA, protein and functional levels. In the mouse, studies at the mRNA level have been performed by reverse transcription polymerase chain reaction (Alonso-Llamazares et al., 1995; Cavalliet al., 1997) and Northern blotting (Garcia-Sainz et al., 1994). They have detectedalpha1A-adrenoceptor mRNA in the heart, lung, liver, spleen, kidney, aorta, adipose tissues and several brain regions, including the cortex and cerebellum.Alpha1B-adrenoceptor mRNA was also detected in all of these tissues, although at somewhat lower abundance in the spleen and adipose tissue.Alpha1D-adrenoceptor mRNA was also detected in all of these tissues, although its presence in liver was seen in one (Alonso-Llamazares et al., 1995) but not another (Cavalliet al., 1997) study. Our data demonstrate thatalpha1-adrenoceptors are detectable at the protein level in murine tissues by [3H]prazosin binding, with the rank order of cerebral cortex > cerebellum > liver > lung > kidney > heart > spleen, with the spleen not exhibiting detectable expression.

Further experiments were designed to investigate possible qualitative species heterogeneity, i.e., to define the subtypes ofalpha1-adrenoceptors in the various murine tissues by using competition binding with various subtype-selective agonists and antagonists and inactivation by chloroethylclonidine. Our results demonstrate that murine liver expresses a homogeneous population of alpha1B-adrenoceptors. Similar results have previously been obtained by other investigators (Garcia-Sainz et al., 1994). Although the situation in the other tissues was somewhat more complex, the competition binding data with most subtype-selective drugs indicate that murine cerebellum and lung also express predominantlyalpha1B-adrenoceptors, whereas murine cerebral cortex and kidney express mixedalpha1A- andalpha1B-adrenoceptors in approximate ratios of 30% to 70% and of 50% to 50%, respectively.

Detection of tissuealpha1D-adrenoceptors at the protein level has long been hampered by the lack of selective antagonists. Recently, BMY 7378 has been introduced as an antagonist that is ∼100-fold selective for alpha1D- overalpha1A- andalpha1B-adrenoceptors (Goetz et al., 1995). In our study, BMY 7378 competition curves in murine liver, cerebral cortex, kidney and lung were steep and monophasic, with affinity estimates 100 times lower than those reported for cloned ratalpha1D-adrenoceptors (Goetz et al., 1995; Yang et al., 1997b). Although a low affinity of BMY 7378 for murinealpha1D-adrenoceptors cannot be excluded, these data indicate that thealpha1D-adrenoceptor protein is absent in the respective murine tissues despite the presence of corresponding mRNA (Alonso-Llamazares et al., 1995; Cavalli et al., 1997). Althoughalpha1D-adrenoceptors have been detected at the protein level in rat aorta (Deng et al., 1996), our present data are similar to those found in several rat tissues (Denget al., 1996; Yang et al., 1997b) and the human prostate (Michel et al., 1996), where noalpha1D-adrenoceptor protein was detected in radioligand binding studies despite the presence of corresponding mRNA. The consistency of this finding across three different species indicates that alpha1D-adrenoceptor mRNA may not be efficiently translated into a functional protein and/or that the functional protein is rapidly degraded. Studies with cloned humanalpha1D-adrenoceptors in our laboratory have indicated that agonist exposure causes lessalpha1D-adrenoceptor down-regulation, if any, compared with alpha1A- oralpha1B-adrenoceptors (Yang et al., 1997a). This makes inefficient translation a more likely explanation of low protein abundance than does rapid degradation ofalpha1D-adrenoceptors. Further investigations into this question were beyond the scope of the present paper. The remaining part of the discussion will therefore focus on thealpha1A- andalpha1B-adrenoceptor distribution at the protein level in murine tissues.

The present study has obtained consistent affinity estimates for all compounds across all investigated tissues. These estimates are in good agreement with those we have previously reported after using identical methods for the human prostate (Michel et al., 1996), bovine cerebral cortex (Büscher et al., 1996), a variety of rat (Büscher et al., 1996; Michel et al., 1993a) and guinea pig (Büscher et al., 1996) tissues and for cloned rat and bovinealpha1-adrenoceptors (Michel and Insel, 1994; Chess-Williams et al., 1996; Michel et al., 1996). They are also in good agreement with those previously reported for murine hepatic alpha1B-adrenoceptors by other investigators (Garcia-Sainz et al., 1994). Taking into consideration the large interlaboratory variation in reported drug affinities of alpha1-adrenoceptor subtypes, they are also in the mainstream of affinities reported by other investigators for cloned subtypes (Michel et al., 1995). Thus, at least for the presently investigated drugs, murinealpha1-adrenoceptors appear to have a qualitatively similar pharmacological profile as those in rats, guinea pigs, cows and humans.

The utility of mice as model systems depends not only on the similarity of pharmacological receptor profiles but also on the quantitative and qualitative receptor subtype expression in comparison to other species. Our data indicate that the interspecies variation in tissue-specificalpha1-adrenoceptor subtype expression appears to be considerably greater than that of drug affinities at a given subtype. For example, on a quantitative level, we were unable to detect quantifiable alpha1-adrenoceptor binding in murine spleen, whereas splenic expression ofalpha1-adrenoceptors (mostly of thealpha1B-subtype) is well detected in rats (Michel et al., 1993a) and guinea pigs (Büscheret al., 1996). On a qualitative level, the murine liver exhibited a homogeneous population ofalpha1B-adrenoceptors in the present and a previous study (Garcia-Sainz et al., 1994). Although homogeneous populations ofalpha1B-adrenoceptors are also found in rat (Garcia-Sainz et al., 1994; Büscher et al., 1996) and hamster (Garcia-Sainz et al., 1994) liver, homogeneous populations ofalpha1A-adrenoceptors were reported for human (Garcia-Sainz et al., 1995) and guinea pig (Garcia-Sainz and Romer-Avila, 1993) liver and mixedalpha1A/alpha1B-adrenoceptor populations for rabbit liver (Torres-Marquez et al., 1991;Garcia-Sainz et al., 1992; Taddei et al., 1993). In the cerebral cortex, the contribution ofalpha1A-adrenoceptors in mice (vide infra) appears to be similar to that in guinea pigs (Büscheret al., 1996), lower than that in rats (Han and Minneman, 1991; Michel et al., 1993a) and humans (Gross et al., 1989), and much lower than that in cows (Büscheret al., 1996). In the kidney, similar densities ofalpha1A- andalpha1B-adrenoceptors were seen in mice (vide infra) and rats (Han et al., 1990; Michelet al., 1993a), butalpha1B-adrenoceptors were predominantly reported in guinea pigs (Büscher et al., 1996). Thus, considerable interspecies heterogeneity exists with regard to the quantitative and qualitative expression ofalpha1-adrenoceptor subtypes in several tissues. Therefore, the identification of a givenalpha1-adrenoceptor subtype as a mediator of a specific functional response in mice may not always correctly predict which subtype mediates this response in other species. This caveat may also limit the utility of knockout mice as model systems.

In some murine tissues, tamsulosin behaved differently from otheralpha1A-selective drugs. Specifically, tamsulosin differentiated two types of sites in the murine lung and cerebellum that were not discriminated by a variety of other drugs,e.g., (+)-niguldipine, despite their greater subtype selectivity. We have previously reported a similar aberrant behavior of tamsulosin in guinea pigs (Büscher et al., 1996). Thus, in guinea pig kidney, a panel of seven subtype-selective drugs indicated a homogeneous population ofalpha1B-adrenoceptors, whereas tamsulosin detected 39% high-affinity sites. In the guinea pig cerebral cortex, these seven subtype-selective drugs detectedalpha1A- andalpha1B-adrenoceptors in approximately a 25% to 75% ratio, whereas tamsulosin detected a 51% to 49% ratio. This raises the possibility that the high- and low-affinity sites for tamsulosin in some murine and guinea pig tissues may not correspond toalpha1A- andalpha1B-adrenoceptors, respectively. However, it should be noted that noradrenaline behaved similarly to tamsulosin in the guinea pig tissues (Büscher et al., 1996), and 5-methylurapidil behaved similarly to tamsulosin in murine lung (vide infra). To clarify the nature of tamsulosin low-affinity sites in some murine tissues, we have performed competition binding experiments for tamsulosin and, as a reference compound, for (+)-niguldipine in the cerebral cortex ofalpha1B-adrenoceptor knockout mice. According to our previous studies, this tissue is a homogeneous source of murine alpha1A-adrenoceptors (Cavalliet al., 1997). Because tamsulosin and (+)-niguldipine detected only high-affinity sites in the cerebral cortex ofalpha1B-adrenoceptor knockout mice, these data suggest that high- and low-affinity sites for tamsulosin indeed correspond to alpha1A- andalpha1B-adrenoceptors, respectively. The reason for quantitatively different ratios of the two subtypes as determined with tamsulosin versus otheralpha1A-selective drugs is not fully clear, but it should be noted that [3H]tamsulosin may label different numbers ofalpha1-adrenoceptors than [3H]prazosin in some tissues and even with cloned subtypes (Yazawa et al., 1992; Michel and Goepel, 1998). Thus, it is possible thatalpha1-adrenoceptor ligands from distinct chemical classes labelalpha1-adrenoceptor subtypes in a quantitatively different manner. Although elucidation of the reasons for such differences was beyond the scope of this paper, these data indicate that percentages of high- and low-affinity sites for a singlealpha1A-selective compound should be interpreted only with great care with regard to the quantitative presence of these subtypes.

The alkylating agent chloroethylclonidine has historically been useful in establishing alpha1-adrenoceptor heterogeneity (Han et al., 1987). In some studies on rat tissues, the proportion of chloroethylclonidine-sensitive and -insensitive sites has corresponded well with the proportion ofalpha1B- andalpha1A-adrenoceptors, respectively, as determined by competition binding with subtype-selective antagonists (Minneman et al., 1988; Wilson and Minneman, 1989; Fenget al., 1991). In contrast, chloroethylclonidine readily alkylates cloned alpha1A-adrenoceptors (Hanet al., 1995; Schwinn et al., 1995; Büscheret al., 1996; Hirasawa et al., 1997), and this has contributed to the initial failure to correctly classify the cloned bovine alpha1A-adrenoceptor (Schwinn et al., 1990). Meanwhile, it has become clear that the ability of chloroethylclonidine to alkylatealpha1-adrenoceptor subtypes depends on the incubation temperature and duration and on the osmolarity of the buffer system. When all of these conditions are kept constant, some subtype selectivity can indeed be seen in the alkylating effects of chloroethylclonidine (Schwinn et al., 1995). We have previously demonstrated that incubation with 10 μM chloroethylclonidine for 30 min at 37°C in a low-osmolarity buffer is necessary and sufficient for full inactivation of rat tissuealpha1B-adrenoceptor but has little effect on rat tissue alpha1A-adrenoceptors (Michelet al., 1993b). Under these conditions, chloroethylclonidine powerfully inactivated alpha1-adrenoceptors in all murine tissues of the present study. In this respect, no major differences were seen between tissues with considerablealpha1A-adrenoceptor fractions (cerebral cortex and kidney) and those without (liver). This is in agreement with some studies on cloned alpha1-adrenoceptor subtypes (Hirasawa et al., 1997) but in clear contrast to the aforementioned studies on rat tissuealpha1-adrenoceptor subtypes. Therefore, we have investigated the concentration-dependent inactivation of murine tissue alpha1B-adrenoceptors (liver) andalpha1A-adrenoceptors (cerebral cortex from alpha1B-adrenoceptor knockout mice; Cavalli et al., 1997). Although these experiments demonstrated statistically significant discrimination of subtypes by chloroethylclonidine, the difference was only small, and tissuealpha1A-adrenoceptors were readily inactivated by 10 μM chloroethylclonidine. These observations are similar to those made under identical conditions in guinea pigs (Büscher et al., 1996). Thus, our study highlights potential pitfalls in the use of chloroethylclonidine for the identification of alpha1-adrenoceptor subtypes and gives further evidence for a very cautious interpretation of data obtained with chloroethylclonidine.

In summary, the present study has detected and characterizedalpha1-adrenoceptor subtypes at the protein level in a variety of murine tissues. The overall pharmacological profile of murine alpha1-adrenoceptor subtypes appears to be similar to that in other species. However, the quantitative and qualitativealpha1-adrenoceptor subtype expression in a given tissue appears to differ considerably between species. Therefore, the utility of mice for the identification ofalpha1-adrenoceptor subtypes mediating a given response may be limited.

Footnotes

  • Send reprint requests to: Dr. Martin C. Michel, Nephrology Laboratory IG 1, Klinikum, 45122 Essen, Germany. E-mailmartin.michel{at}uni-essen.de

  • ↵1 This work was supported in part by grants from the Deutsche Forschungsgemeinschaft and Fonds National Suisse de la Recherche Scientifique (3100–051043).

  • Abbreviations:
    BMY 7378
    (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride)
    SB 216469
    N-(3-[4-(2-methoxyphenyl)-l-piperazinyl]propyl)-3-methyl)-4-oxo-2-phenyl-4H-l-benzopyran-8-carboxamide monomethanesulfonate monohydrate
    IC50
    median inhibitory concentration
    • Received November 10, 1997.
    • Accepted April 29, 1998.

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Murine Alpha 1-Adrenoceptor Subtypes. I. Radioligand Binding Studies

Ming Yang, Jens Reese, Susanna Cotecchia and Martin C. Michel
Journal of Pharmacology and Experimental Therapeutics August 1, 1998, 286 (2) 841-847;
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