Classification of α1-adrenoceptor subtypes

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Classification of α1-adrenoceptor subtypes

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  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 pharmacolo- gical techniques, and three distinct cDNAs encoding gl-adrenoceptor subtypes have been cloned. The pro- file of an increasing number of subtype-selective com- pounds at cloned and endogenous receptors recently has facilitated alignment between cloned and pharma- cologically defined gl-adrenoceptor subtypes. Thus, ~a-adrenoceptors (previously designated ~1¢), ~ib-ad- renoceptors and ~a-adrenoceptors (previously desig- nated ~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 addition- ally 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 classifica- tion have not always been straightforward and have generated considerable confusion within and even more outside the group of e~-adrenoceptor investiga- tors. More recent evidence, however, has allowed reso- lution of most of these problems. Therefore, we will describe the current concept of ~a-adrenoceptor sub- type 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 norad- renaline. 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 srcinally sug- gested 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 [3HI 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 phen- tolamine (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 antagon- ists. Numerous other compounds with differing degrees of selectivity and/or various other types of ancillary properties have been introduced since, e.g. metho- xamine (Tsujimoto et al. 1989) or oxymetazoline (Hanft et al. 1989). Based on the above pharmacological tools a num- ber of receptor binding and functional studies have characterized el-adrenoceptor subtypes in various tis- sues and cell lines (Table 1). While the definition of subtypes may have somewhat shifted with the intro- duction of new compounds, two types of pharmacolo- gically defined ~a-adrenoceptors were usually detected (Lomasney et al. 1991a; Bylund 1992). One of them has high affinity for the agonists methoxamine and oxy- metazoline and the antagonists WB 4104, phen- tolamine, 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 cere- bral cortex, hippocampus, kidney and heart. While the cqB-adrenoceptor has srcinally 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 AHllll0A (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 re- vealed by receptor cloning studies. The first cq-ad- renoceptor 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 en- coded a protein with high affinity for WB 4101 and phentolamine; the expression product was somewhat sensitive to alkylation by CEC and corresponding mRNA was srcinally not detected by Northern blot analysis in cq-adrenoceptor-containing rat tissues (Schwinn et al. 1990). This clone was srcinally desig- nated 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 corres- pond to the pharmacologically-defined ~A-adrenocep- tor, 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~-ad- renoceptors but the cloned and pharmacologically-de- fined ~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 detec- ted in a variety of tissues known to contain ~lB-ad- renoceptors, and is the only ~l-adrenoceptor subtype mRNA detected in tissues known to express a homo- geneous 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 srcinally desig- nated ~c is the molecular correlate of the cqA-ad- renoceptor, 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, phen- tolamine 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 rea- sons: 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-selec- tive 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 transmembrane 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-ad- renoceptors 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 het- erogeneity 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 iden- tical 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 demon- strated 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 srcinally 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 pharma- cologically-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-adrenocep- tor (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 cat- echolamines 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 con- taining ~A- and ~B-adrenoceptors (e.g. rat cerebral cortex) except for rat kidney (Michel et al. 1993a, 1993b). This relative selectivity of endogeneous cat- echolamines 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 ~-adreno- 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 sub- types, and the general problem of resolving competi- tion 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 com- pounds. Thus, following chloroethylclonidine treat- ment 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 identifica- tion of a tissue or cell line natively expressing a homo- geneous 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-methyl- urapidil, (+)-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 sel- ectivity for ~B- vs. ~aA- or em-adrenoceptors has been suggested for risperidone (Sleight et al. 1993) or AHlll0A (King et alo 1994) but these observations have not yet been confirmed. Similarly, antagonists with high selectivity for ~-relative 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 ~-adrenocep- tor 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 selecti- vity for ~IB- or ~Z1D- adrenoceptors are desirable. Are additional ~l adrenoceptor subtypes likely to exist? A fourth ~-adrenoceptor subtype has not been detec- ted 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 prob- lems associated with presently available pharmacolo- gical tools. Therefore, the evidence in favour of a fourth subtype rests on functional data. In organ bath studies with smooth muscle preparations considerable vari- ation 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 func- tional site with low prazosin affinity represents an addi- tional ~l-adrenoceptor subtype which they have desig- nated ~ (Muramatsu et al. 1990; Ohmura et al. 1992). Whether this site represents an additional ~l-ad- renoceptor 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 phar- macological 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 sub- type 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 proto- types 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-de- fined ~-adrenoceptor subtypes. Thus, ~d-adrenocep- tor 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
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