The α_1L-adrenoceptor is an alternative phenotype of the α_1A-adrenoceptor

Abstract

Despite over two decades of research, the molecular identity of the α_1L-adrenoceptor phenotype has remained elusive. In this issue of the BJP, Gray et al. (2008) provide persuasive evidence that the in vivo α_1L-adrenoceptor phenotype requires the expression of the α_1A-adrenoceptor gene. They have shown that in mice lacking the functional α_1A-adrenoceptor gene, α_1L-mediated responses to noradrenaline in prostate smooth muscle are substantially attenuated. These findings support earlier evidence that the α_1L-adrenoceptor profile represents a functional phenotype of the α_1A-adrenoceptor gene product, but additional cell background-dependent factors must act in concert with the α_1A-adrenoceptor protein to determine whether an α_1L- or a classical α_1A-adrenoceptor profile is expressed. The challenge remains to establish the nature of these cellular factors and the mechanism(s) by which they influence G-protein-coupled receptor pharmacology.

In the ‘post-genomic' era, much attention has been focused on the study of the physiological roles and endogenous ligands for previously undiscovered G-protein-coupled receptors (GPCRs), identified from genomic sequencing (so-called ‘deorphanization'). In contrast, the α₁-adrenoceptor field has struggled with the opposite conundrum—a pharmacologically defined receptor phenotype, which has resisted molecular definition. Genes have been identified for three isoforms of the α₁-adrenoceptor (termed α_1A, α_1B and α_1D), but the fourth α₁-adrenoceptor phenotype, α_1L-adrenoceptor, has until now been defined purely on the basis of a characteristically low affinity for a number of selective antagonists, including prazosin (Guimaraes and Moura, 2001). However, this phenotype is of physiological significance, as the α_1L-adrenoceptor profile has been identified in a variety of tissues, across a number of different species (see Guimaraes and Moura, 2001), where it regulates smooth muscle contractility in the vasculature and the lower urinary tract. It may also be of clinical relevance, as α₁-adrenoceptor antagonists such as tamsulosin are a frontline therapy for benign prostatic hyperplasia, where they effectively and selectively relax prostatic smooth muscle, providing symptomatic relief for BPH patients (Milani and Djavan, 2005).

It has previously been proposed that the α_1L phenotype may represent an alternative conformational state of the α_1A-adrenoceptor gene product (Ford et al., 1997). When recombinantly expressed in Chinese hamster ovary cells, the α_1A-adrenoceptor exhibited a classical α_1A-adrenoceptor profile in radioligand-binding assays in membrane homogenates, but in [³H]-inositol phosphate accumulation assays in intact cells, a number of antagonists (including prazosin and 5-methylurapidil) displayed lower affinities, consistent with the pharmacological profile of the α_1L-adrenoceptor (Ford et al., 1997). In addition, whereas the native α₁-adrenoceptor expressed in rat prostate smooth muscle exhibited an α_1A profile in membrane radioligand-binding assays, the functional (contractile) phenotype in the same tissue was that of an α_1L-adrenoceptor (Hiraoka et al., 1999). These (and other) early studies, therefore, pointed to the α_1L-adrenoceptor being the functional manifestation of the α_1A-adrenoceptor gene product. However, the dependence upon assay conditions (that is, functional assays in intact cells/tissues versus radioligand-binding assays in membrane homogenates) of the observed phenotype, allied to the fact that functional α_1A profiles can be observed in some tissues (see Guimaraes and Moura, 2001 and references therein) has confounded attempts to establish the relationship between the α_1A- and α_1L-adrenoceptors.

An analogous situation to the atypical pharmacological profile of the α_1L-adrenoceptor is that of the putative β₄-adrenoceptor, a phenotype defined by resistance to classical β-adrenoceptor antagonists and activation by so-called ‘non-conventional partial agonists' (Kaumann, 1989). The molecular identity of this phenotype (as a novel ‘state' of the β₁-adrenoceptor) was identified by the use of ‘knockout' mice lacking combinations of β-adrenoceptors (see Granneman, 2001 and references therein). In this issue of the BJP, Gray et al. (2008) apply a similar approach to provide the first definitive evidence that the manifestation of the α_1L-adrenoceptor phenotype (at least, in mouse prostate smooth muscle) is dependent upon the expression of the α_1A-adrenoceptor gene product. Using a range of antagonists known to display selectivity between the α_1A- and α_1L-adrenoceptor profiles, the authors have previously characterized the noradrenaline-mediated contraction of mouse prostate smooth muscle as being mediated by an α_1L-adrenoceptor (Gray and Ventura, 2006). In the present study, Gray et al. (2008) utilized ‘knockout' mice lacking a functional α_1A-adrenoceptor gene (Rokosh and Simpson, 2002), to investigate the role of this gene in the observed α_1L in vivo phenotype. They found that responses to noradrenaline were attenuated by approximately 80% in prostates from mice homozygous for the disrupted α_1A-adrenoceptor gene, compared with wild-type mice, providing strong evidence that the expression of the α_1L-adrenoceptor in mouse prostate smooth muscle requires the presence of a functional α_1A-adrenoceptor gene (Gray et al., 2008).

In addition, the authors also examined contractile responses to electrical field stimulation, an experimental paradigm more closely resembling physiological stimulation. This contraction was partially inhibited by prazosin and the contraction to high-frequency stimulation was approximately 30% smaller in mice lacking the functional α_1A-adrenoceptor than in wild-type mice (Gray et al., 2008). Importantly, the residual contraction (most probably mediated by non-adrenergic, non-cholinergic transmitters) was insensitive to prazosin, indicating that all of the α_1A/L-adrenoceptor-mediated contraction was lost in the absence of the α_1A gene. The case might have been strengthened if the authors had demonstrated that the adrenergic component of the electrical field-stimulated contraction was mediated by α_1L-adrenoceptors, as the authors themselves acknowledge that the receptors mediating responses to nerve stimulation could differ from those mediating the response to exogenous noradrenaline. However, together with their findings with exogenously applied noradrenaline, these data provide the strongest evidence thus far that the α_1A-adrenoceptor gene is essential for the generation of the α_1L-adrenoceptor phenotype.

Providing that the dependence of the α_1L phenotype upon α_1A-adrenoceptor gene expression is universally applicable (across all species/tissues where the α_1L phenotype has been identified), the next question to address is what determines whether an α_1A-adrenoceptor exhibits an α_1L- or a classical α_1A-adrenoceptor phenotype? The fact that functional responses in certain tissues display a classical α_1A-adrenoceptor profile (see Guimaraes and Moura, 2001) suggests that the α_1L-phenotype is not simply the default functional profile of the α_1A-adrenoceptor gene product, raising the possibility that tissue-dependent cellular factors may govern the observed phenotype (Nelson and Challiss, 2007). It is well established that the cellular environment can influence GPCR signalling and agonist pharmacology, but the traditional view that the antagonist pharmacology is independent on the cellular context may also need to be reevaluated (Nelson and Challiss, 2007).

Evidence has recently been presented that the intact cellular environment is important for the manifestation of the in vivo α_1L-adrenoceptor phenotype (Morishima et al., 2007, 2008). These studies have shown that both α_1A- and α_1L-adrenoceptor populations can be distinguished in radioligand-binding assays in intact tissue segments, but that upon tissue homogenization and membrane preparation, the α_1L-adrenoceptors are either degraded or converted to α_1A-adrenoceptors (Morishima et al., 2007, 2008). Clarification of what is happening to the α_1L-adrenoceptor population upon its isolation in membrane homogenates might provide valuable clues as to the cellular factor(s) responsible for shaping the pharmacological profile of the α_1A-adrenoceptor gene product. Numerous mechanisms for generating phenotypic pharmacological profiles of GPCRs have been identified (see Nelson and Challiss, 2007 and references therein) and as our appreciation of the complexity of GPCR signalling advances, so does the list of possibilities. The identification of the α_1L-adrenoceptor as an alternative phenotype of the α_1A-adrenoceptor represents a significant advance in our understanding of this phenomenon and will hopefully provide a springboard for future progress in elucidating the mechanisms underlying these distinct phenotypes.

Abbreviations

BPH

benign prostatic hyperplasia

GPCR

G-protein-coupled receptor