Classification of α1-adrenoceptor subtypes

Naunyn-Schmiedeberg's Arch Pharmacol (1995) 352:1 10 © Springer-Verlag 1995

Martin C. Michel • Barry Kenny • Debra A. Schwinn

Classification of l-adrenoceptor subtypes

Received: 2 February 1995/Accepted: 23 February 1995

Abstract Two gl-adrenoceptor subtypes (~IA and ~IB) have been detected in various tissues by pharmacological techniques, and three distinct cDNAs encoding gl-adrenoceptor subtypes have been cloned. The profile of an increasing number of subtype-selective compounds at cloned and endogenous receptors recently has facilitated alignment between cloned and pharmacologically defined gl-adrenoceptor subtypes. Thus, ~a-adrenoceptors (previously designated ~1¢), ~ib-adrenoceptors and ~a-adrenoceptors (previously designated ~la, ~la or ~la/d) are now recognized. Since the ~a-adrenoceptor shares characteristics with both ~A- and ~B-adrenoceptors, tissues previously reported to express glA- and/or ~iB-adrenoceptors may additionally contain ~a-adrenoceptors. This article reviews the features of all three subtypes and discusses possible pitfalls in their pharmacological identification.

Key words CqA-Adrenoceptor • ~ lB-Adrenoceptor - C~li)-Adrenoceptor • Nomenclature

current concepts of ~l-adrenoceptor subtype classification have not always been straightforward and have generated considerable confusion within and even more outside the group of e~-adrenoceptor investigators. More recent evidence, however, has allowed reso- lution of most of these problems. Therefore, we will describe the current concept of ~a-adrenoceptor subtype classification and developments leading to this concept; additionally we will highlight problems which have occurred along the way and at least partly still persist. The discussion of such problems may also be helpful for investigators trying to classify other receptor families since many of these pitfalls do not appear to be specific for ~l-adrenoceptor subtypes. This article will mainly focus on data obtained by radioligand binding and recombinant DNA technology as they appear most powerful for receptor subtype classification. Functional aspects of cq-adrenoceptor heterogeneity have previously been reviewed elsewhere (Minneman 1988; Garcia-Sainz 1993; Ruffolo and Hieble 1994).

Introduction

et-Adrenoceptors mediate many of the physiological effects of the catecholamines adrenaline and noradrenaline. In recent years it has become clear that el- adrenoceptors are not a homogeneous entity but rather constitute a distinct subfamily within the overall family of adrenoceptors. The developments leadings to the

M. C. Michel ( ~ ) Department of Medicine, Nephrology laboratory JG1, Klinikum, University of Essen, Hufelandstrasse 55, D-45122 Essen, Germany

B. Kenny Department of Discovery Biology, Pfizer Central Research, Kent, UK

D. A. Schwinn Departments of Anesthesiology, Pharmacology and Surgery, Duke University Medical Center, Durham, North Carolina, USA

Historical aspects

Heterogeneity of cq-adrenoceptors was originally suggested by Morrow and Creese (1986) on the basis of radioligand binding studies in rat brain. In these initial experiments they found that phentolamine and WB 4101 competed for [ 3 H I prazosin binding with shallow and biphasic curves whereas prazosin, indoramine and dihydroergocryptine exhibited steep and monophasic competition curves; they designated the high and low affinity site for WB 4101 and phentolamine as C~IA- and elB-adrenoceptors 1, respectively. In 1987 Han et al. demonstrated that chloroethylclonidine (CEC)

l In this paper we will refer to pharmacologically-defined tissue receptor subtypes by upper case subscripts (e.g. elA) and to cloned subtypes by lower case subscripts (e.g. ~la)

2

Table 1 Drug affinities at rat tissue cq-adrenoceptor subtypes ~IA ~IB

5-Methylurapidil 8.8 + 0.6 (7.7 9.3) 7.1 _+ 0.5 (6.0 7.8) ( + )-Niguldipine 9.7 + 0.8(8.6 10.7) 7.2 _+ 0.6(6.3 8.1) Oxymetazoline 8.4 _+ 0.4 (7.7-9.0) 6.4 _+ 0.3 (5.8-6.8) Phentolamine 8.5 _+ 0.7 (7.3-9.3) 7.3 _+ 0.5 (6.6 8.1) WB 4101 9.7 _+ 0.5(8.6-10.3) 8.4 _+ 0.4(7.8 9.3) Inactivation by chloroethylclonidine No Yes

Data are mean _+ SD and range of -log Ki values as determined in various rat tissues by numerous investigators (Hanft and Gross 1989; Boer et al. 1989; Hanft et al. 1989; Minneman et al. 1988; Michel et al. 1989, 1993a, 1994a; Han and Minneman 1991). CqA-Adrenoceptor affinities were taken from high affinity sites in rat cerebral cortex, hippocampus, vas deferens, kidney and heart, cqB-adrenoceptors from liver, spleen and low affÉnity sites in cerebral cortex hippocampus, vas deferens, kidney and heart data

irreversibly binds to and inactivities ~B-adrenoceptors in a variety of rat tissues (Han et al. 1987). Thereafter, the serotonin 5-HT1A agonist 5-methylurapidil was shown to preferentially bind to ~A-adrenoceptors with a selectivity exceeding that of WB 4101 or phentolamine (Gross et al. 1988; Hanft and Gross 1989). Finally, the dihydropyridine-type Ca 2 ÷ entry blocker (+)-niguldipine was also shown to be selective for ~lA-adrenoceptors in radioligand binding studies (Boer et al. 1989; Gross et al. 1989), and this drug still is one of the most subtype-selective cq-adrenoceptor antagonists. Numerous other compounds with differing degrees of selectivity and/or various other types of ancillary properties have been introduced since, e.g. methoxamine (Tsujimoto et al. 1989) or oxymetazoline (Hanft et al. 1989).

Based on the above pharmacological tools a number of receptor binding and functional studies have characterized el-adrenoceptor subtypes in various tissues and cell lines (Table 1). While the definition of subtypes may have somewhat shifted with the introduction of new compounds, two types of pharmacologically defined ~a-adrenoceptors were usually detected (Lomasney et al. 1991a; Bylund 1992). One of them has high affinity for the agonists methoxamine and oxymetazoline and the antagonists WB 4104, phentolamine, 5-methylurapidil, and ( + )-niguldipine but is resistant to alkylation by CEC (Table 1); this subtype has been named the ~A-adrenoceptor. Homogeneous populations of ~lA-adrenoceptors have been reported in rat submaxillary gland (Michel et al. 1989), rabbit liver (Garcia-Sainz et al. 1992; Taddei et al. 1993) and guinea pig liver (Garcia-Sainz et al. 1992; Garcial-Sainz and Romer-Avila 1993; but see below); it coexists with other cq-adrenoceptor subtypes in rat cerebral cortex, hippocampus, vas deferens, kidney, and heart. Another subtype has considerably lower affinity for the above competitive agonists and antagonists but is sensitive to alkylation and inactivation by CEC (Table 1); this subtype has been named the elB-adrenoceptor. Homo- geneous populations of cqB-adrenoceptors have been found in rat liver and spleen (Han and Minneman 1991; Garcia-Sainz et al. 1992; Michel et al. 1993a); it co-

exists with other cq-adrenoceptor subtypes in rat cerebral cortex, hippocampus, kidney and heart. While the cqB-adrenoceptor has originally been defined by its low affinity for various drugs and its sensitivity towards alkylation by CEC (Tables 1 and 2), more recent data indicate that competitive antagonists with selectivity for cqB-adrenoceptors such as risperidone (Sleight et al. 1993) or A H l l l l 0 A (King et al. 1994) may also exist.

Thus, two types of criteria have been used to differ- entiate el-adrenoceptor subtypes pharmacologically, i.e. affinity for competitively acting drugs, as well as sensitivity to the alkylating effects of CEC. In initial studies it appeared that etA- and cqB-adrenoceptors may selectively couple to specific signaling pathways, i.e. that C~A-adrenoceptors activate influx of extracellu- lar Ca 2+, preferably through L-type Ca 2+ channels, and that ~lB-adrenoceptors activate a phospholipase C yielding formation of inositol phosphates and diacyl- glycerol (Minneman 1988). More recent data, however, suggest that the usefulness of this approach may be of limited value (Bylund et al. 1994).

~-Adrenoceptor heterogeneity has also been revealed by receptor cloning studies. The first cq-adrenoceptor cDNA was cloned from the smooth muscle cell-derived hamster cell line DDTa-MF2 and encoded a protein which has low affinity for all known e~A- adrenoceptor-selective drugs and is readily alkylated by CEC; hence it was named the ~b subtype (Cotecchia et al. 1988). Thereafter, a homologous cDNA was cloned from a bovine brain cDNA library which encoded a protein with high affinity for WB 4101 and phentolamine; the expression product was somewhat sensitive to alkylation by CEC and corresponding mRNA was originally not detected by Northern blot analysis in cq-adrenoceptor-containing rat tissues (Schwinn et al. 1990). This clone was originally designated cqo (Schwinn et al. 1990) but is now recognized to encode an CqA-adrenoceptor (see below). Finally, a third cNDA was cloned by homology screening from a rat cerebral cortex cDNA library; due to its high affinity for some ~A-adrenoceptor-selective drugs and to presence of corresponding mRNA in many e~A-adrenoceptor-containing rat tissues this clone was

Table 2 Drug affinities at cloned ~l-adrenoceptor subtypes

~la ~lb ~ld

Previous names ~1o ~lb ~la~ (~la/d

5-Methylurapidil 8.63 _+ 0.32 6.97 _+ 0.50 7.31 _+ 0.66 (8.17 9.30) (5.98 8.21) (6.21 8.26)

Methoxamine 4.29 _+ 0.75 3.36 _+ 0.67 4.33 _+ 0.61 (3.13-5.54) (2.67-5.24) (3.18-5.27)

( + )-Niguldipiue 8.57 -- 1.12 6.84 _+ 0.75 6.55 _+ 0.43 (7.10 10.22) (5.77 8.10) (5.96 7.34)

Noradrenaline 5.00 _+ 0.54 5.35 _+ 0.42 6.53 _+ 0.49 (4.39 6.48) (4.76-6.03) (5.81 7.39)

Oxymetazoline 7.53 _+ 0.43 6.50 _+ 0.48 5.81 _+ 0.51 (6.70 8.22) (5.36 6.96) (4.43 6.22)

Phentolamine 8.17 _+ 0.49 7.20 _+ 0.36 7.48 _+ 0.42 (7.47-9.02) (6.47-7.82) (6.86-8.21)

Prazosin 9.52 +_ 0.38 9.79 _+ 0.38 9.63 _+ 0.40 (8.66-10.22) (9.25-10.46) (8.71-10.40)

WB 4101 9.32 _+ 0.32 8.01 _+ 0.44 8.83 _+ 0.42 (8.86 10.09) (7.07 8.80) (7.85-9.37)

Data are mean _+ SD and range of-log Ki of 9-17 published studies using rat, human, hamster and bovine clones expressed transiently or stably in a variety of cell lines as assessed by either [3H]prazosin or [125I]BE 2254 as the radioligand (Schwinn et al. 1990, 1991, 1995; Lomasney et al. 1991b; Perez et al. 1991, 1994; Schwinn and Lomas- ney 1992; Ramarao et al. 1992; Hirasawa et al. 1993; Kenny et al. 1994; Michel and Insel 1994; Forray et al. 1994; Weinberg et al. 1994; Laz et al. 1994; Faure et al. 1994; Minneman et al. 1994; Goetz et al. 1994; Testa et al. 1995)

initially designated 0(la (Lomasney et al. 1991b); in subsequent studies, however, it became clear soon that the expression product of this cDNA does not correspond to the pharmacologically-defined ~A-adrenoceptor, and designations such as aid (Perez et al. 1991) or ~z,/d (Schwinn and Lomasney 1992) were introduced; the designation ~ a has now been adopted by the IUPHAR nomenclature committee (see below) based on the proposal of Ford et al. (1994).

Thus, both pharmacological and receptor cloning studies have clearly suggested heterogeneity of c~-adrenoceptors but the cloned and pharmacologically-defined ~l-adrenoceptor subtypes were not easily aligned. Therefore, many subsequent studies were designed to further the alignment of cloned and pharmacologically- defined Czl-adrenoceptor subtypes and/or to identify and clone additional subtypes. These attempts have been successful and agreement on the classification of ~l-adrenoceptor subtypes is now emerging.

Present status of ~l-adrenoceptor subtype classification

The first cq-adrenoceptor subtype cDNA clone was obtained from a hamster smooth muscle cell line (Cotecchia et al. 1988); species homologs of this clone have been obtained from rat (Lomasney et al. 1991b; Voigt et al. 1990) and human sources (Forray et al.

1994; Weinberg et al. 1994; Schwinn et al. 1995). In man the corresponding gene structure has also been elucid- ated (Ramarao et al. 1992). Immediately it became clear that these clones indeed represent an cqB-adrenoceptor for three reasons (Table 2): Firstly, their expression products have low affÉnity for all known ~ lA-ad - renoceptor-selective drugs (Cotecchia et al. 1988; Schwinn and Lomasney 1992; Kenny et al. 1994; Michel and Insel 1994; Weinberg et al. 1994; Forray et al. 1994; Perez et al. 1994; Testa et al. 1995; Faure et al. 1994; Schwinn et al. 1995) (Table 2). Secondly, these expression products are readily alkylated by CEC, and in a direct comparison with the receptors encoded by the other clones are the most sensitive towards alkylation (Perez et al. 1991, 1994; Laz et al. 1994; Forray et al. 1994; Schwinn et al. 1995). Thirdly, mRNA corresponding to these clones is readily detected in a variety of tissues known to contain ~lB-adrenoceptors, and is the only ~l-adrenoceptor subtype mRNA detected in tissues known to express a homogeneous population of ~l~-adrenoceptors such as rat liver (Lomasney et al. 1991b; Rokosh et al. 1994; Price et al. 1994a; Faure et al. 1994). The tissue distribution of such mRNA has now also been mapped for a variety of human tissues (Price et al. 1994b; Weinberg et al. 1994). Taken together these data clearly demonstrate that the clones initially designated ~lb indeed encode for an ~lB-adrenoceptor.

Which if any of the cloned subtypes encodes for the ~A-adrenoceptor remained unclear in initial studies, but it is now accepted that the clone originally designated ~c is the molecular correlate of the cqA-adrenoceptor, and hence has been redesignated ~la by the IUPHAR nomenclature committee (Table 3). Although the clone isolated from bovine brain encodes a protein with high affinity for most e~A-adrenoceptor-selective drugs including oxymetazoline, WB 4101, phentolamine and 5-methylurapidil (Schwinn et al. 1990; Perez et al. 1991, 1994; Schwinn and Lomasney 1992; Kenny et al. 1994; Michel and Insel 1994; Faure et al. 1994; Testa et al. 1995; Table 2), it was initially not thought to encode the Cqa-adrenoceptor for three reasons: Firstly, its mRNA was not detectable in any rat tissue by Northern blot analysis (Schwinn et al. 1990, 1991). Secondly, the expression product of the bovine ~la clone was somewhat sensitive to alkylation by CEC (Schwinn et al. 1990). Thirdly, in initial studies it had very low affinity for the most ~lA-adrenoceptor-selective drug, (+)-niguldipine (Schwinn and Lomasney 1992). All these problems have now been resolved, at least partly due to the cloning of the rat (Laz et al. 1994; Perez et al. 1994) and human homologs (Hirasawa et al. 1993; Forray et al. 1994; Weinberg et al. 1994; Schwinn et al. 1995) of the cqa-adrenoceptor. Thus, expressed rat and human ~la-adrenoceptor clones have high affinity for ( + )-niguldipine (Laz et al. 1994; Forray et al. 1994; Weinberg et al. 1994; Perez et al. 1994), and more recent studies have also detected high ( + )-niguldipine

4

Table 3 Characteristics of cq-adrenoceptor subtypes

~IA ~IB ~ID

Previous names ~ c (Z1A, O~IA/D

Structure 7 TM 7 TM 7 TM 466 aa 515 aa 560 aa

Human chromosome 8 5 20

Selective drugs ( +)-Niguldipine and 5- No highly selective compound has Noradrenaline vs. c~la and ~IB Methylurapidil vs. ~IB and cqD been established consistently methoxamine and WB 4101 vs. ~

CEC-sensitivity + / - + + + + +

Structural information refers to the human clones (7TM: 7 putative t ransmembrane domains; aa: amino acids)

affinity for the bovine ~t,-adrenoceptor (Testa et al. 1995; Schwinn et al. 1995). They may undergo some alkylation by CEC but quantitatively this is much less than those of e~b- or e~a-adrenoceptors (Forray et al. 1994; Laz et al. 1994; Perez et al. 1994; Schwinn et al. 1995). Finally, ~,-adrenoceptor mRNA has now been detected in various rat (Alonso-Llamazares et al. 1993; Rokosh et al. 1994; Laz et al. 1994; Price et al. 1994a; Faure et al. 1994; Perez et al. 1994) and human tissues (Hirasawa et al. 1993; Price et al. 1993, 1994b; Wein- berg et al. 1994) by RNAse protection assay, RT-PCR, in situ hybridization and in contrast to initial studies by Northern blot analysis; it is the most abundant or even only detectable ~a-adrenoceptor mRNA form in tissues known to contain predominantly or solely e~A-adrenoceptors such as rat submaxillary gland (Rokosh et al. 1994; Faure et al. 1994) or human prostate (Price et al. 1993; Weinberg et al. 1994). For these reasons it is now accepted that the ela-adrenoceptor clone formerly designated as ~c indeed is the molecular correlate of the ~tA-adrenoceptor.

The understanding of e~-adrenoceptor subtype heterogeneity has been made difficult for some time by the cloning of a third subtype (Lomasney et al. 1991b), which shared some properties with e~A- and some with etB-adrenoceptors. Confusion was furthered by dis- agreement on the nomenclature since apparently identical clones were initially designated ~ , (Lomasney et al. 1991b) and ~ld (Perez et al. 1991), and later ~l,/a (Schwinn and Lomasney 1992). A human species homolog of this clone has also been isolated (Bruno et al. 1991; Forray et al. 1994; Weinberg et al. 1994; Schwinn et al. 1995). These clones were initially be- lieved to encode an elA-adrenoceptor for three reasons: Firstly, expression products of these clones had high affinity for the e~A-adrenoceptor-selective antagonist WB 4101 (Lomasney et al. 1991b; Perez et al. 1991). Secondly, its mRNA was found in many rat tissues known to express elA-adrenoceptors (Lomasney et al. 1991b). Thirdly, guinea pig liver had been demonstrated to express a homogeneous population of ea~- like adrenoceptors (Garcia-Sainz et al. 1992; Garcia- Sainz and Romer-Avila 1993), and only a rat probe corresponding to this new subtype but not a hamster

~lb or bovine ~la probe hybridized to guinea pig liver mRNA in Northern blots (Garcia-Sainz et al. 1992). Therefore, it had been proposed that this clone which is now designated eld might encode an e~A-adrenoceptor (Lomasney et al. 1991b; Garcia-Sainz et al. 1992). This belief was also supported by the initial failure to recog- nize the bovine clone originally designated as ~o as the molecular correlate of the etA-adrenoceptor. On the other hand, it became clear soon that the e~d-Clone is different from the ~lA-adrenoceptor in many respects. The expressed e~a-adrenoceptor had only low affinity for a number of ~aA-adrenoceptor-selective drugs such as 5-methylurapidil, oxymetazoline or (+)-nigul- pidipine (Perez et al. 1991, 1994; Schwinn and Lomas- ney 1992; Forray et al. 1994; Michel and Insel 1994; Weinberg et al. 1994; Laz et al. 1994; Faure et al. 1994; Testa et al. 1995; Schwinn et al. 1995; Table 2). Thus, drug affinities at the expressed ~a-adrenoceptor did not correlate as well with those determined at pharmacologically-defined ~aA-adrenoceptors as those of the expressed bovine ela-clone when tested under identical conditions (Michel and Insel 1994; Faure et al. 1994; Testa et al. 1995). Moreover, in a direct comparison the expression product of the ~a-adrenoceptor clone was considerably more sensitive towards alkylation of chloroethylclonidine than the e~,-adrenoceptor, and it was almost as sensitive as the expressed e~b-adrenoceptor (Forray et al. 1994; Laz et al. 1994; Perez et al. 1994; Schwinn et al. 1995). The argument that a rat ~la-but not a bovine el~-adrenoceptor probe hybridized to mRNA prepared from guinea pig liver (Garcia-Sainz et al. 1992), may not be valid due to problems in cross-species hybridization; in this context it should be noted that a rat (Alonso-Llamazares et al. 1993; Rokosh et al. 1994; Laz et al. 1994; Price et al. 1994a; Perez et al. 1994) but not a bovine el,-adrenoceptor probe (Schwinn et al. 1990,1991) hybridized to mRNA in a number of e~A-adrenoceptor-containing rat tissues in Northern blot analysis. Finally, the catecholamines noradrenaline and adrenaline have higher affinity for the ela-adrenoceptor compared to other cloned ~-adrenoceptor subtypes in most studies (Lomasney et al. 1991b; Perez et al. 1991, 1994; Forray et al. 1994; Michel and Insel 1994; Laz et al. 1994;

Minneman et al. 1994; Testa et al. 1995; Table 2) but do not discriminate between subtypes in most tissues containing ~A- and ~B-adrenoceptors (e.g. rat cerebral cortex) except for rat kidney (Michel et al. 1993a, 1993b). This relative selectivity of endogeneous catecholamines for the %d-adrenoceptor is intriguing but its physiological relevance remains to be investigated.

Taken together the above data demonstrate that the ~d-adrenoceptor clone indeed represents a third subtype which is distinct from ~A- and ~ - a d r e n o - ceptors. This opens the question why such a third subtype had not previously been detected in native tissues or cell lines. Given the similarities of ~d- with both e~A- and el~-adrenoceptors, the low degree of selectivity of many compounds to distinguish the subtypes, and the general problem of resolving competition binding curves into more than two components experimentally, this failure is not too surprising. On the other hand it should be noted that the percentage of ~lA- and ~a~-adrenoceptors determined by different compounds varied within a tissue in some studies (Hart and Minneman 1991; Michel et al. 1993a), which may reflect differential recognition of eaa-adrenoceptors as either ~IA- or ~-adrenoceptors by the various compounds. Thus, following chloroethylclonidine treatment two binding sites remain in rat kidney which can be discriminated by a number of compounds (Michel et al. 1993a), and one of these may correspond to the ~d-adrenoceptor (Michel and Insel 1994). Further identification of tissue ela-adrenoceptors will probably require the introduction of drugs with high selectivity (see below). Preliminary data indicate that BMY 7378 may be an antagonist with an approximately 100-fold selectivity for ~d-relative to other ~-adrenoceptor subtypes (Saussy et al. 1994). Moreover, the identification of a tissue or cell line natively expressing a homogeneous population of e~a-adrenoceptors would be quite useful.

Taken together three subtypes of ~-adrenoceptors are now recognized and designated ~A, ~B and ~m (Table 3; (Ford et al. 1994)). Drugs with selectivity for ~A- over elB-adrenoceptors include 5-methylurapidil, (+)-niguldipine, and WB 4101. e~A-Ad- renoceptors are best differentiated from em-adreno- receptors by their high affinity for ( + )-nigulpidine or oxymetazoline. Among competitively acting drugs selectivity for ~B- vs. ~aA- or em-adrenoceptors has been suggested for risperidone (Sleight et al. 1993) or A H l l l 0 A (King et alo 1994) but these observations have not yet been confirmed. Similarly, antagonists with high selectivity for ~ - re la t ive to e~A- or ~ - adrenoceptors have not been established (except for preliminary data on BMY 7378 (Saussy et al. 1994)), but the endogeneous catecholamines adrenaline and noradrenaline may be approximately 10-fold selective. While chloroethylclonidine inactivates ~-adrenoceptor subtypes with a rank order of ea~ _> ~1i~ > ~A, many problems exist with the use of this tool (see below).

More selective drugs, in particular those with selectivity for ~IB- or ~Z1D- adrenoceptors are desirable.

Are additional ~l-adrenoceptor subtypes likely to exist?

A fourth ~-adrenoceptor subtype has not been detected at the cDNA or gene level despite considerable efforts by various laboratories, but absence of proof is not proof of absence. Previous claims for the detection of additional ~l-adrenoceptor subtypes in radioligand binding studies may stem, at least in part, from problems associated with presently available pharmacological tools. Therefore, the evidence in favour of a fourth subtype rests on functional data. In organ bath studies with smooth muscle preparations considerable variation in the functional potency of prazosin has been reported. Since prazosin has similar affinity for all cloned ~-adrenoceptor subtypes (Table 2), Muramatsu and colleagues have repeatedly proposed that the functional site with low prazosin affinity represents an additional ~l-adrenoceptor subtype which they have designated ~ (Muramatsu et al. 1990; Ohmura et al. 1992). Whether this site represents an additional ~l-adrenoceptor subtype remains to be established.

The existence of additional subtypes can also be postulated from theoretical considerations. Thus, be- cause introns are present in ~l-adrenoceptor genes, splice variants are possible. In analogy to the dopamine receptors such splice variants might have distinct pharmacological characteristics (O'Dowd 1993). Whether this possibility holds true for ~l-adrenoceptors remains to be investigated.

Pitfalls in ~-adrenoceptor suMype classification

The path of scientific discovery into ~l-adrenoceptor subtypes since the first report of cloning of such a subtype has been winded and too often confusing to in- siders and bystanders. In the following we will discuss some of the problems which have occurred along the way in the hope that recognition of such pitfalls may help investigators in other fields to avoid them.

Receptor subtypes in general and ~l-adrenoceptor subtypes in particular display tissue-specific expression. Therefore, certain tissues have been taken as prototypes of e~-adrenoceptor subtypes. For example, the rat submaxillary gland and liver have been taken as prototypes of ~A- and ~B-adrenoceptors, respectively (Michel et al. 1989; Han and Minneman 1991). There- fore, tissue distribution of mRNA has been used as a criterium to align cloned and pharmacologically-defined ~-adrenoceptor subtypes. Thus, ~d-adrenoceptor mRNA was initially found in many rat tissues known to contain e~A-adrenoceptors using a rat ~d- adrenoceptor probe (Lomasney et al. 1991b), while mRNA corresponding the ~-adrenoceptor could not

be detected in rat tissues using the bovine ~a-adrenoceptor probe (Schwinn et al. 1990,1991). More- over, an apparently homogeneous population of e~A- like adrenoceptors was detected in guinea pig liver but only a rat ~a- but not a bovine ~la-adrenoceptor probe hybridized to mRNA from this tissue (Garcia-Sainz et al. 1992). These data highlight two problems with the mRNA approach: Firstly, mRNA expression may be an indicator of corresponding protein expression in many cases but it is not very reliable, particularly on a quantitative level. For example, fil-adrenoceptor mRNA is five times as abundant as fi2-adrenoceptor mRNA in human liver whereas only fi2- but not fia-adrenoceptors are detectable at the protein level in radioligand binding studies (Arner et al. 1990). Secondly, the failure of bovine ela-adrenoceptor probes to hybridize to rat Cqa-adrenoceptor mRNA under stringent conditions (Schwinn et al. 1990, 1991) demonstrates that even mi- nor species differences at the cDNA level may considerably hamper cross-species hybridization. Thus, mRNA tissue distribution can be taken as an indicator of receptor identity if other means of identification are not available but its power should not be overestimated.

In the original studies leading to the concept of al-adrenoceptor subtype heterogeneity it had been postulated that a~A- and ~lB-adrenoceptors can be distinguished based on their differential signalling mecha- nisms (Minneman 1988). Thus, it was proposed that CqA-adrenoceptors promote Ca 2÷ influx predominantly via a dihydropyridine-sensitive channel whereas ~B-adrenoceptors promote Ca 2÷ mobilization from intracellular stores secondary to activation of inositol phosphate formation. While this may be true in many model systems, examples can be found where CqA-adrenoceptors partly (Wilson and Minneman 1990a; Michel et al. 1993b) or fully (Knowlton et al. 1993) mediate the inositol phosphate response in rat kidney or neonatal cardiomyocytes, respectively, two tissues expressing both subtypes. The bovine cqa- and hamster Cqb-adrenoceptor both couple to inositol phosphate formation upon expression in HeLa cells, and the ela- adrenoceptor may do so even more efficiently (Schwinn et al. 1991). On the other hand, ~B-adrenoceptors have been found to activate Ca 2 + influx through a dihydropyridine-sensitive channel in rat medullary thyroid carcinoma 6 23 cells (Esbenshade et al. 1994). In other studies it has been found that a pertussis toxin-sensitive G-protein mediates CqA- but not a,B-adrenoceptor signaling in primary cultures from rat brain (Wilson and Minneman 1990b), whereas the ~B-adrenoceptor in rat liver may act via a pertussis toxin-sensitive G-protein (Butta et al. 1993). Following heterologous expression of cloned ~l-adrenoceptor subtypes a given subtype may mediate different signaling responses via pertussis toxin-sensitive and -insensitive G-proteins (Perez et al. 1993). Thus, preferred signalling pathways do not appear to be reliable to discriminate cq-adrenoceptor subtypes.

The pharmacological identification of receptors and their subtypes classically depends on the affinities of various selective drugs, with regard to G-protein- coupled receptors preferably antagonists. On the other hand it is well known that affinity estimates obtained in various laboratories under different conditions may vary somewhat. It should be expected that many of these differences are due to variation among the model systems used. Thus, most of these differences should disappear when cloned subtypes are used. Unfortu- nately, this expectation has not turned into reality in the ~l-adrenoceptor field. While most subtype-selective drugs have selectivity factors of less than 100, reported affinities frequently vary between investigators by the same magnitude or even more (Tables 1 and 2). Ob- served affinity estimates are apparently not related to the species from which the ~l-adrenoceptor subtype has been cloned (with the possible exception of ( + )- niguldipine), the expression technique (i.e. transient vs. stable), the expression system (e.g. COS-7, rat-1 or HeLa cells), or the radioligand used (i.e. [3H~prazosin vs. [125I] BE 2254). Thus, the reasons for these discrep- ancies are unclear but they may be a reflection of inappropriate experimental conditions. For example, ligand depletion can be encountered in binding assays with small assay volumes, notably with [3Hlprazosin, which can lead to inaccurate estimations of affinity. Long incubation times may be necessary with some compounds to reach equilibrium and obtain reliable affinity estimates, especially in multicellular preparations where diffusion may be slow, e.g. for isolated organs in functional assays. In addition, the affinity of the selective compound ( + )-niguldipine can be dependent on the level of assay protein since the drug is highly lipophilic (Boer et al. 1989).

For many of the confounding factors influencing apparent affinity estimates it could be expected that absolute number might vary whereas affinity ratios between subtypes should do so less. However, even affinity ratios can vary considerably between laboratories (Michel and Insel 1994; Testa et al. 1995). Thus, before a given clone is considered to represent a certain known receptor subtype, a range of subtype-selective compounds should be studied by a number of indepen- dent investigators.

Another issue is that of species specific receptor subtype characteristics. As discussed above cross-species hybridization at the mRNA level may be mislead- ing. Species problems can also occur at the protein level. Thus, the affinity of ( + )-niguldipine at the expressed cloned bovine cqa-adrenoceptor was initially reported to be 100-1000 fold lower (Schwinn and Lomasney 1992) than that found in native tissues (Table 1) or with its cloned rat or human homologs (Table 2). This observation casted doubt on the identity of the bovine clone, and in light of other data at least indicated considerable species heterogeneity in drug affinities. More recent data from the original (Schwinn

et al. 1995) and other investigators (Testa et al. 1995), however, indicate that ( + )-niguldipine also has high affinity for bovine ~a-adrenoceptors. Moreover, preliminary data from one of our laboratories indicate that an ~l-adrenoceptor subtype with high ( + )-niguldipine affinity is detectable in bovine brain (Biischer, Heeks and Michel, unpublished observations). Thus, the original report on low ( + )-niguldipine affinity of bovine %a-adrenoceptors may have been erroneous. Neverthe- less other adrenoceptor subtypes also indicate that species differences in pharmacological profile can be quite large even when differences at the amino acid level are fairly small. For example, a single amino acid switch in the mouse ~2A-adrenoceptor can markedly affect its affinity for rauwolscine (Link et al. 1992). Thus, preferably all subtypes should be cloned from the same species before a clear alignment between cloned and tissue receptor subtypes can be made.

In the case of ~l-adrenoceptor not only competitively acting antagonists and agonists have been used as pharmacological tools for subtype definition, but the irreversibly acting CEC has been used as well. CEC was originally introduced as an irreversible ~e-adrenoceptor agonist (Leclerc et al. 1980). Several years later CEC was reintroduced as a drug which selectively inactivates the ~l-adrenoceptor subtype with low affinity for WB 4101, i.e. the ~n-adrenoceptor (Han et al. 1987). Ori- ginally CEC has been used as a rather qualitative tool, i.e. inactivating ~IB- adrenoceptors without affecting elA-adrenoceptors. More recent studies, however, have seriously questioned this use of CEC for several reasons: Firstly, experiments with cloned ~-adrenoceptor subtypes indicate that sensitivity towards alkylation by CEC is a quantitative rather than qualitative feature. Thus, alkylation appears to increase for any given subtype with CEC concentration, incubation time, and temperature (Perez et al. 1991, 1994; Schwinn et al. 1995). With this knowledge a comparison of CEC sensi- tivities among laboratories is difficult, since the experimental conditions used by various investigators differ with regard to concentration (3-100 ~tmol/1), incubation time (10 m i n - 20 h) and temperature (4-37 ° C). Secondly, in competition binding assays CEC binds equally well to ~A- and ~t~-adrenoceptors although it only inactivates e~-adrenoceptors under these conditions (Michel et al. 1993c, 1994a); these data imply that CEC could be a reversible, possibly competitive antagonist at ~lA-adrenoceptors. If this is true, CEC could be of limited value under in vivo conditions where CEC remaining after ~.l~-adrenoceptor alkylation may not successfully be washed out and thus also block 7~A- adrenoceptors. Finally, CEC can also act on 7e-adrenoceptors where it behaves as an irreversible agonists at ~eA- and 7~c-adrenoceptors and binds reversibly to ~z~-adrenoceptors (Michel et al. 1993c; Biiltmann and Starke 1993; Nunes and Guimaraes 1993). Thus, CEC may have sympatholytic effects which are related to presynaptic ~2-adrenoceptor agonism rather than post-

synaptic ~lB-adrenoceptor inactivation (Vargas et al. 1994). Therefore, we conclude that CEC may be useful to discriminate ~l-adrenoceptor subtypes, but its use requires careful controls with regard to the assay conditions used in order to achieve selective ~lB-adrenoceptor inactivation; an effect on certain ~2-adrenoceptor subtypes or a reversible affect on ~lA-adrenoceptors should also be ruled out. Since ~d-adrenoceptor sensitivity towards alkylation by CEC is intermediate compared to ~a- and ~lb-adrenoceptors (see above), it is possible that ~l-adrenoceptors remaining after CEC treatment reflect a mixed ~la- and ~ld-adrenoceptor population.

Radioligand binding studies on tissue ~-adrenoceptor subtypes have mostly used [3H]prazosin as the radioligand. While prazosin does not discriminate among the cloned ~-adrenoceptor subtypes (Table 2), some functional data have suggested the existence of an ~-adrenoceptor subtype with low affinity ( > 1 nmol/1) for prazosin (Muramatsu et al. 1990; Oh- mura et al. 1992). Although some attempts have been made to label such ~l-adrenoceptors with high concen- trations of l-3H]prazosin, they were not very successful since a very high proportion of non-specific binding resulted. Alternate radioligands for c~-adrenoceptors such as [-125I]BE 2254 also have apparently similar affinity for ~-adrenoceptor subtypes (Hanft et al. 1989; Michel et al. 1994a), but have not been tested in functional models where prazosin has low affinity. Thus, the available literature on radioligand binding studies with e~-adrenoceptors may be biased towards those subtypes with high prazosin affinity.

As an overall conclusion from the above it appears that presently available pharmacological tools are not sufficiently subtype-selective to allow a clear discrimi- nation of ~-adrenoceptor subtypes in all settings. Moreover, the two most selective tools, 5-methylurapidil and (+)-niguldipine, have ancillary properties, i.e. 5-HT1A receptor agonism and L-type Ca 2÷ channel antagonism respectively, which limit their usefulness in physiological studies. Thus, the development of more selective ligands is highly necessary to ease ~l-adrenoceptor subtype classification.

Future perspectives

Based on the above it appears that the mystery of ~l-adrenoceptor subtype classification is now mostly solved. Accordingly an alignment between cloned and pharmacologically-defined native subtypes has been agreed upon by the IUPHAR nomenclature committee (Table 3), and will be published soon. This alignment has adopted the nomenclature proposed by Ford et al. (1994) recognizing the ~lA-, ~IB- and ~lD-adrenoceptors. Nevertheless a number of important questions remain to be answered. For example, with regard to

possible species differences in subtype characteristics it remains to be established whether ~l,-adrenoceptor mRNA is detectable in guinea pig liver when appropri- ate probes are used or whether indeed an apparently homogenous elA-adrenoceptor population corres- ponds to the presence of ~a-adrenoceptor mRNA only (Garcia-Sainz et al. 1992). Further confirmation on the pharmacological characteristics of the bovine eaA-adrenoceptor particularly with regard to ( + )-niguldipine also appears desirable.

A more important problem is the physiological relevance of el-adrenoceptor subtypes. This could in- volve the mediation of different functions within a tissue, activation under different conditions and/or different regulation patterns. Generally the functional identification of ~l-adrenoceptor subtypes has been more difficult than that in radioligand binding studies, but some progress has been made. In this respect some data indicate for example that in cultured neonatal rat cardiomyocytes the hypertrophic response may be mediated by an elA-adrenoceptors (Knowlton et al. 1993) whereas the inotropic response in adult rat heart muscle strips occurs mostly via an e~B-adrenoceptor (Michel et al. 1994b). Functional correlates of the elO- adrenoceptor have not unequivocally been identified. Given the higher affinity of endogenous catecholamines for aid- compared to other el-adrenoceptors (see above), it will be interesting to learn whether cqD- adrenoceptors are preferentially activated in tissues co- expressing this and other subtypes.

The clearer identification of functional cq-adrenoceptor subtypes will require the development of more selective pharmacological tools. The possibility that such compounds may have fewer side effects than established non-subtype selective cq-adrenoceptor antagonists will certainly stimulate the development of such drugs in the near future.

Acknowledgements Work in the authors' laboratories is supported by grants from NIH (HL49103 and HL 02490 to DAS), Deutsche Forschungsgemeinschaft (Ph 43/1-3 to MCM), Pfizer Central Re- search (to DAS) and Yamanouchi Pharmaceuticals (to MCM and DAS).

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Classification of α1-adrenoceptor subtypes

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