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In the α1-adrenoceptors, three distinct subtypes (α1A-, α1B- and α1D-adrenoceptors; nomenclature follows Alexander et al., 2008) have been cloned and are known to be involved in various physiological functions (Lomasney et al., 1991; Hieble et al., 1995; Zhong and Minneman, 1999; Michelotti et al., 2000). The three classical α1-adrenoceptors have high (subnanomolar) affinity for prazosin, a prototypic, selective α1-adrenoceptor antagonist, but show distinct pharmacological profiles for several antagonists. For example, silodosin, 5-methylurapidil and RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethamine hydrochloride) are selective for α1A-adrenoceptors, and BMY-7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione-dihydrochloride) is selective for α1D-adrenoceptors (Lomasney et al., 1991; Hieble et al., 1995; Ford et al., 1996; Murata et al., 1999; Piao et al., 2000). Tamsulosin is a potent antagonist for the three α1-adrenoceptor subtypes or shows slightly lower affinity for the α1B subtype than for the other two subtypes (Ford et al., 1996; Morishima et al., 2008).
In addition to these classical α1-adrenoceptors, the presence of another subtype (the putative α1L-adrenoceptor) has been proposed (Flavahan and Vanhoutte, 1986; Muramatsu et al., 1990; Ford et al., 1996). The α1L-adrenoceptor shows a unique pharmacological profile: low affinity for prazosin, 5-methylurapidil, RS-17053 and BMY 7378, but high affinity for silodosin and tamsulosin (Muramatsu et al., 1995, 1998; Ford et al., 1996; Murata et al., 1999). The α1L-adrenoceptors have been identified primarily by functional studies: rabbit thoracic aorta (Oshita et al., 1993), rabbit iris (Nakamura et al., 1999; Suzuki et al., 2002), rat small mesenteric arteries (Stam et al., 1999), canine subcutaneous arteries (Argyle and McGrath, 2000), rat and human vas deferens (Ohmura et al., 1992; Amobi et al., 2002), and rabbit, rat, mouse and human prostate (Ford et al., 1996; Van der Graaf et al., 1997; Hiraoka et al., 1999; Gray and Ventura, 2006; Morishima et al., 2007a; Su et al., 2008). However, the gene corresponding to the α1L-adrenoceptor has not yet been cloned, even though many trials of candidate genes have been carried out, including splicing variants of α1-adrenoceptor genes and heterodimeric expression of different subtypes (Ramsay et al., 2004). Subsequently, it has been considered that the α1L-adrenoceptor may be a functional phenotype of the α1A-adrenoceptor because the functional studies with recombinant α1A-adrenoceptors have revealed a relatively low affinity for prazosin (Ford et al., 1997; Daniels et al., 1999).
Recently, we have demonstrated in radioligand binding studies that α1L-adrenoceptors occur as a distinct entity from α1A-, α1B- and α1D-adrenoceptors (Hiraizumi-Hiraoka et al., 2004; Morishima et al., 2007a, 2008). However, the α1L-adrenoceptors were detected only under intact tissue conditions and the pharmacological profile converted to that of the α1A-adrenoceptor after homogenization (Morishima et al., 2008; Su et al., 2008). This line of evidence strongly suggests that the α1L-adrenoceptor may be a phenotypic subtype of α1-adrenoceptors (probably the α1A-adrenoceptor) rather than a genetically different subtype (Su et al., 2008).
The aims of this study were to identify α1L-adrenoceptors in mice by radioligand binding and bioassay approaches and to examine the effects of gene knockout of the classical α1-adrenoceptor subtypes. The present results show that binding and functional profiles of α1L-adrenoceptors can be clearly detected in wild-type (WT) mice, but that the α1L-adrenoceptor is selectively abolished in α1A-adrenoceptor gene knockout (AKO) mice. A part of this study was presented in the Satellite Meetings (Pharmacology of Adrenoceptors, Beijing, China, 2006) of the 15th International Congress of Pharmacology and the 80th Annual Meeting of The Japanese Pharmacological Society (Morishima et al., 2007b).
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[3H]-Silodosin has very high apparent affinities for both α1A-adrenoceptor and α1L-adrenoceptor subtypes (Murata et al., 1999; Morishima et al., 2008). In recent radioligand binding studies with [3H]-silodosin, it has been demonstrated that α1L-adrenoceptors are pharmacologically distinct from the three classical α1-adrenoceptors (α1A, α1B and α1D) in the intact segments of the rat cerebral cortex (Morishima et al., 2008), rabbit ear artery (Hiraizumi-Hiraoka et al., 2004) and human and rabbit prostate (Morishima et al., 2007a; Su et al., 2008). In the present binding study with [3H]-silodosin, the α1L-adrenoceptor was recognized as a distinct site showing low affinity for prazosin, RS-17053 and BMY 7378, but high affinity for silodosin and tamsulosin in the intact tissue segments of the cerebral cortex, vas deferens and prostate of WT mouse, along with the α1A-adrenoceptor, which showed high affinity for prazosin, RS-17053, silodosin and tamsulosin. The same results were also obtained in BKO and DKO mice, whereas the [3H]-silodosin binding sites disappeared in AKO mice. Furthermore, the functional studies with mouse vas deferens and prostate revealed that the α1-adrenoceptors mediating contractile responses to noradrenaline showed an α1L-adrenoceptor profile and that the contractions were specifically abolished in AKO mice, but not in BKO and DKO mice. Adrenergic contractions in vas deferens and prostate have been reported through α1L-adrenoceptors in many mammalian species including mice (Ohmura et al., 1992; Hiraoka et al., 1999; Ford et al., 1996; Van der Graaf et al., 1997; Amobi et al., 2002; Gray and Ventura, 2006; Morishima et al., 2007a; Su et al., 2008). Thus, the present results strongly suggest that α1L-adrenoceptors occur and function independently from other α1-adrenoceptor subtypes, but that the expression of the α1L-adrenoceptor is closely related to that of the α1A-adrenoceptor gene, and that the α1B-adrenoceptor and the α1D-adrenoceptor are not involved in manifestation of the α1L-adrenoceptor phenotype.
More recently, we reported in a study with the rat cerebral cortex that the α1L-adrenoceptor could be detected in the intact segments, but that the pharmacological profile disappeared after homogenization (Morishima et al., 2008). The same phenomenon was observed in this study with WT mouse cortex. Interestingly, in both species, the total binding density of [3H]-silodosin in the tissue segments of cerebral cortex was equal to the density of α1A-adrenoceptors detected in the membrane preparations (Figure 1b). Because contamination with α1B-adrenoceptors and α1D-adrenoceptors is negligible in the present [3H]-silodosin binding sites (Murata et al., 1999; Morishima et al., 2008), it seemed that the pharmacological profile of the α1L-adrenoceptors converted to that of the α1A-adrenoceptors by homogenization without a loss of yield. Moreover, the α1A-adrenoceptors in the membrane preparations were abolished in AKO mice. These results show that both α1A-adrenoceptor and α1L-adrenoceptor are derived from the same α1A-adrenoceptor gene, and further suggest that α1L-adrenoceptor is a pharmacological phenotype of α1A-adrenoceptor (Su et al., 2008).
[3H]-prazosin also bound to the intact segments of WT mouse cortex with a high affinity and the binding sites were divided into two components by silodosin but not by BMY 17053. A similar population of silodosin high- and low-affinity sites and the same densities were observed in DKO mouse cortex (Figure 1c). However, sites for silodosin were selectively abolished in AKO mice (no high-affinity sites) or BKO mice (no low-affinity sites), resulting in a complete loss of the corresponding subtypes. These results show that [3H]-prazosin at the concentrations used in this experiment can selectively recognize α1A-adrenoceptors and α1B-adrenoceptors in the intact segments of WT and DKO mouse cortices, as reported in the rat cerebral cortex (Morrow and Creese, 1986; Morishima et al., 2008).
The α1A-, α1B- and α1L-adrenoceptor subtypes were distinctly identified in the [3H]-silodosin and [3H]-prazosin binding sites in WT mouse, whereas in AKO mice, the α1A and α1L subtypes were specifically abolished and the α1B subtype selectively disappeared in BKO mice. Thus, we conclude that no quantitative/qualitative compensation by other subtypes of the same α1-adrenoceptor group (for example, upregulation or supersensitivity) is caused in knockout mice, which supports previous in vivo and in vitro studies with AKO, BKO and DKO mice (Cavalli et al., 1997; Rokosh and Simpson, 2002; Tanoue et al., 2002; Simpson, 2006). This conclusion also accounts for a complete loss of α1L-adrenoceptor-mediated responses in the vas deferens and prostate of AKO mice and no significant shift of pEC50 values for noradrenaline between WT and BKO or DKO mouse prostate. It can be noted that the expression of each α1-adrenoceptor subtype is differentially regulated in cardiac myocytes (Rokosh et al., 1996).
In contrast to AKO mouse vas deferens, a small but significant contraction was elicited by noradrenaline in AKO mouse prostate (Figure 7a). This residual contraction was insensitive to silodosin (1 nM, a specific concentration for α1A and α1L-adrenoceptors) and BMY 17053 (10 nM, a specific concentration for α1D-adrenoceptor) but potently inhibited by prazosin (1 nM, a specific concentration for α1A-, α1B-, or α1D-adrenoceptors) (Figure 7b). Therefore, a part of the contractile response to noradrenaline in mouse prostate may be mediated through α1B-adrenoceptors, although the major component is still α1L-adrenoceptor-mediated.
This study clearly revealed that two different phenotypes (α1A and α1L-adrenoceptors) originate from a single α1A-adrenoceptor gene, yet no evidence explains why multiple phenotypes are produced from a single gene. However, it is interesting, in this context, to note the history of the β4-adrenoceptor. Like α1L-adrenoceptors, β4-adrenoceptors were originally identified as functional receptors resistant to several β-adrenoceptor antagonists, such as propranolol (Kaumann and Molenaar, 1996; Sarsero et al., 1998). However, the β4-adrenoceptor was absent after β1-adrenoceptor gene knockout (Kaumann et al., 2001). At present, the β1-adrenoceptor has been considered to have two distinct binding sites within the same receptor, orthosteric and allosteric sites with high and low affinity for catecholamines or propranolol (Konkar et al., 2000; Baker and Hill, 2007). The β4-adrenoceptor reflects the pharmacological profiles of an allosteric site of β1-adrenoceptors with low affinity for propranolol. Thus, it may be possible that the α1L phenotype represents the presence of an allosteric site in the α1A-adrenoceptor.
Another interesting finding in this study was that, even though α1L-adrenoceptors occurred as a separate entity, independent of α1A-adrenoceptors, in the intact segments, the pharmacological profile completely changed to that of classical α1A-adrenoceptors upon homogenization. Such a change in profile upon homogenization was not seen in β4-adrenoceptors (Kaumann et al., 2001; Joseph et al., 2003). Thus, if the α1L-adrenoceptor is an allosteric site variant of the α1A-adrenoceptor, it is likely that a more drastic conformational change is caused by homogenization in α1A-adrenoceptors than in β1-adrenoceptors, resulting in a greater change in profile. Alternatively, the α1L-adrenoceptor may be a phenotype constructed with some associated proteins and the construct may be disrupted by homogenization. This has been demonstrated in the receptor for calcitonin gene-related peptide, in which different phenotypes are produced by association with different receptor-modifying proteins (McLatchie et al., 1998; Poyner et al., 2002). Moreover, the α1L-adrenoceptor might be constructed under a particular environment with specific parameters, such as microdomains, relation to the cytoskeleton and extracellular ion environment, without involving an associated protein. Recently, it has been suggested that pharmacological properties may not necessarily remain constant between tissues expressing the same receptors or between different assay conditions (Kenakin et al., 1995; Muramatsu et al., 2005; Nelson and Challiss, 2007). Thus, the molecular mechanisms underlying the expression of the α1L-adrenoceptor phenotype should be explored in future studies.
In summary, this study using knockout mice demonstrated that α1L-adrenoceptors can be identified as binding and functional entities in mice, and strongly suggested that two distinct phenotypes (α1A-adrenoceptor and α1L-adrenoceptor) are derived from the same α1A-adrenoceptor gene.