I. Introduction

Top Previous Next References

"The nerves controlling the blood-vessels that supplied his face functioned so well that the skin, robbed of all its blood, went quite cold, the nose looked peaked, and the hollows beneath the young eyes were lead-couloured as any corpse's. And the Sympathicus caused his heart, Hans Castorp's heart, to thump, in such a way that it was impossible to breathe except in gasps; and shivers ran over him, due to the functioning of the sebaceous glands, which, with the hair follicles, erected themselves".Thomas Mann, 1924

The operation of the sympathetic nervous system, especially of its cardiovascular branches, is nowhere in literature described better than in this passage from Thomas Mann's Magic Mountain, that great novel on pre-1914 Europe that the author places in a sanatorium at Davos in the Swiss mountains. Vasoconstriction, tachycardia, and contraction of the musculi arrectorum pilorum are Hans Castorp's autonomic responses when he first addresses his beloved Claudia Chaucat on Walpurgis-Night to borrow a pencil from her. This review probes the mechanisms that noradrenaline, the classical transmitter substance of the sympathetic vasoconstrictor fibers, uses to make blood vessels constrict; probes, in other words, the events that occurred in Hans Castorp when he borrowed the pencil.

Directly or indirectly, the blood vessels are the source of many and serious diseases that affect millions of people. In many respects, vascular physiology and pharmacology have changed dramatically over the last years. The discovery by Furchgott and Zawadzski in 1980 of endothelium-derived relaxing factor (EDRF²) revolutionized our knowledge and placed the endothelium in the center of the physiology and pathophysiology of the vascular tree; the cloning of many receptors brought about a true "Renaissance" in receptor pharmacology (Kenakin, 1997); and the possibility to "knock out" specific genes in experimental animals represents a new and important tool for a detailed study of the adrenoceptors, including those of the vascular system.

The present review aims at updating adrenoceptors in blood vessels, particularly on a functional point of view. Occasionally, some information is derived from nonvascular tissues; however, emphasis is placed on results obtained in blood vessels. Some reviews covering part of the present theme were published in the last few years (Insel, 1996; Strosberg, 1997; Summers et al., 1997; Docherty, 1998; Miller, 1998; Brodde and Michel, 1999; Bünemann et al., 1999; Freissmuth et al., 1999; Guimarães, 1999; Hein, 1999; Zhong and Minemann, 1999; Garcia-Sáinz et al., 2000; Gauthier et al., 2000; Hein, 2000; Kable et al., 2000).

	II. Subclassification of Adrenoceptors

Top Previous Next References

The adrenoceptors are the cell membrane sites through which noradrenaline and adrenaline act as important neurotransmitters and hormones in the periphery and in the central nervous system. The adrenoceptors are targets for many therapeutically important drugs, including those for some cardiovascular diseases, asthma, prostatic hypertrophy, nasal congestion, obesity, and pain.

The first step leading to the discovery of the adrenoceptors was made in the cardiovascular systemthe observation by Dale (1905) that the pressor effect of adrenaline was reversed by ergotoxine into a depressor effect. An explanation for this phenomenon was not apparent until 43 years later! In 1948, Ahlquist noted two patterns in the relative ability of several sympathomimetic agonists to cause pharmacological responses in a series of organs and proposed the division of adrenoceptors into two types,  and . This was subsequently confirmed by the identification of selective antagonists for these two sites: phentolamine and ergotamine for -adrenoceptors; dichloroisoprenaline (Powell and Slater, 1958) and propranolol (Black et al., 1964) for -adrenoceptors. Nineteen years later, it was shown that certain agonists and antagonists could distinguish -adrenoceptor-mediated responses among tissues such as cardiac muscle and bronchial smooth muscle, implying the existence of subtypes of -adrenoceptors (₁ in cardiac muscle and ₂ in the bronchi) (Furchgott 1967, 1972; Lands et al., 1967a,b). Later on, the existence and differential tissue localization of ₁ and ₂ subtypes of -adrenoceptors were discovered and characterized. The existence of subclasses of -adrenoceptors has become evident from the results obtained by Starke and coworkers, who showed that pre- and postjunctional -adrenoceptors differ with respect to the relative potencies of some agonists: low concentrations of clonidine and oxymetazoline selectively activate the prejunctional -adrenoceptors, whereas phenylephrine and methoxamine selectively activate the postjunctional -adrenoceptors (Starke, 1972; Starke et al., 1974, 1975b). Similarly, the relative potency of antagonists supported this differentiation: phenoxybenzamine was about 30 times more potent in blocking postjunctional than prejunctional -adrenoceptors (Dubocovich and Langer, 1974) and yohimbine preferentially blocked prejunctional -adrenoceptors (Starke et al., 1975a). Langer (1974) suggested that -adrenoceptors mediating responses of effector organs should be referred to as ₁ and those mediating a reduction of the transmitter release during nerve stimulation as ₂. Later, it was found that -adrenoceptors pharmacologically very similar to the prejunctional ₂-adrenoceptors are also found postjunctionally. Consequently, the nomenclature of ₁- and ₂-adrenoceptors, depending exclusively on the relative potencies of certain -agonists and antagonists, was accepted (Berthelsen and Pettinger, 1977). In the late 1980s, the development of more selective drugs and the use of molecular cloning technology showed that there are more adrenoceptor subtypes than previously suspected. Nine different subtypes have now been cloned and pharmacologically characterized (Alexander and Peters, 1999).

₁-Adrenoceptors were first divided into two subtypes, _1A and _1B, based on the differential affinity of the receptors for 5-methyl urapidil (5-MU), WB-4101 (Morrow and Creese, 1986; Gross et al., 1988; Hanft and Gross, 1989; Boer et al., 1989) and the irreversible antagonist chloroethylclonidine (Han et al., 1987). _1A-Adrenoceptors showed high affinity for 5-MU and WB-4101 and were insensitive to chloroethylclonidine, and _1B-adrenoceptors were sensitive to CEC and had low affinity for 5-MU and WB-4101. At the present time, a consensus has been reached, such that the subdivision of ₁-adrenoceptors into three subtypes is generally accepted: _1A (formerly _1c; Schwinn et al., 1990), _1B (Cotecchia et al., 1988), and _1D (formerly _1a/d; Lomasney et al., 1991; Perez et al., 1991; Bylund et al., 1994; Ford et al., 1994). In humans, _1A-, _1B-, and _1D-adrenoceptors are encoded by distinct genes located on chromosomes 8, 5, and 20, respectively (Hieble et al., 1995; Michel et al., 1995). Furthermore, human _1A-adrenoceptor heterogeneity comes from the existence of multiple variants that differ in length and sequence of their C-terminal domains (Hirasawa et al., 1995). Additional truncated _1A-adrenoceptor proteins have been reported (Chang et al., 1998). More importantly, no pharmacological or signaling differences were observed on expression of these different splice variants. According to Lattion et al. (1994), they may exhibit differential susceptibility to desensitization. A fourth ₁-adrenoceptor, the so-called _1L-adrenoceptor, has been postulated (Holck et al., 1983; Flavahan and Vanhoutte, 1986a; Muramatsu et al., 1990), based exclusively on pharmacological criteria (e.g., relatively low affinity for prazosin and other antagonists such as RS-17053). This _1L-adrenoceptor seems to mediate constriction of human (Ford et al., 1996) and rabbit (Van der Graaf et al., 1997; Kava et al., 1998) lower urinary tract, guinea pig aorta (Muramatsu et al., 1990), and rat small mesenteric arteries (Stam et al., 1999). However, this hypothetical additional subtype resisted identification by biochemical and/or molecular techniques so far. Recent studies indicate that the _1L-adrenoceptor may not be derived from a distinct gene, but represents a particular, energetically favorable, conformational state of the _1A-adrenoceptor (Ford et al., 1998). Why these two pharmacological phenotypes occur requires further investigation (Ford et al., 1997, 1998).

It is well known that ₁-adrenoceptors are mainly coupled to G_q/11-protein to stimulate phospholipase C activity and that this enzyme promotes the hydrolysis of phosphatidylinositol bisphosphate producing inositol trisphosphate and diacylglycerol. These molecules act as second messengers mediating intracellular Ca²⁺ release from nonmitochondrial pools and activating protein kinase C, respectively (for reviews, see Hein and Kobilka, 1995; Zhong and Minneman, 1999; García-Sáinz et al., 2000). The three cloned ₁-adrenoceptor subtypes have different efficiencies in activating phospholipase C. According to Theroux et al. (1996), the ranking order of coupling efficiency (increase in inositol triphosphate formation and intracellular Ca²⁺) after agonist occupation of recombinant ₁-adrenoceptors expressed in human embryonic kidney 293 cells was: _1A > _1B > _1D. All three ₁-adrenoceptor subtypes can couple to phospholipase C through protein G_q/11, only _1A- and _1B-subtypes couple to protein G₁₄, and only the _1B-subtype couples to protein G₁₆ (Wu et al., 1992). Other studies support that native _1B-adrenoceptors (but not _1A- or _1D-adrenoceptors) can also couple to protein G_o in rat aorta (Gurdal et al., 1997) suggesting a functional role for this coupling. Other signaling pathways have also been shown to be activated by ₁-adrenoceptors: Ca²⁺ influx, arachidonic acid release, phospholipase D activation, and activation of mitogen-activated protein kinase (for a review, see Zhong and Minneman, 1999). Currently, no close relationship can be established between specific subtypes and signaling mechanisms.

₁-Adrenoceptor subtypes are differentially regulated. Although the maximal down-regulation after a prolonged exposure to phenylephrine was similar for _1A- and _1B-adrenoceptors, the threshold concentration of phenylephrine for significant reduction was 100-fold higher for _1A- than for _1B-adrenoceptors. In contrast, phenylephrine up-regulated _1D-adrenoceptors in a time- and concentration-dependent manner (Yang et al., 1999).

It is now clear that there are three subtypes of ₂-adrenoceptors: _2A/D, _2B, and _2C. This subdivision, although primarily based on radioligand binding data, was preceded by results obtained in functional studies and confirmed by molecular cloning. For the _2B- and _2C-adrenoceptors, the pharmacological characteristics are consistent across mammalian species; however, the _2A-adrenoceptor cloned from human and porcine tissue differs slightly in its amino acid composition from the homologous receptor cloned from the rat, mouse, or guinea pig in having a serine residue rather than a cysteine, at the position corresponding to Cys²⁰¹. To the three different genes, four pharmacological subtypes correspond since the Ser²⁰¹ receptor possesses pharmacological properties different from the Cys²⁰¹ receptor, and the two have been distinguished as _2A (e.g., humans) and as _2D (e.g., rodents) (Bylund et al., 1992; Starke et al., 1995; Trendelenburg et al., 1996; Paiva et al., 1997; Guimarães et al., 1998). These two orthologs will be simply referred to as _2A/D, unless some distinction between them has to be made. In humans, the genes coding for _2A-, _2B-, and _2C-adrenoceptors are localized in chromosomes 10, 2, and 4, respectively (Regan et al., 1988; Lomasney et al., 1990; Weinshank et al., 1990).

Pharmacologically it is well known that the different -adrenoceptor antagonists possess different potency/affinity for the different ₂-adrenoceptor subtypes: prazosin for example, has relatively high affinity for _2B- and _2C-adrenoceptors and very low affinity for _2A- and _2D-adrenoceptors (Latifpour et al., 1982; Nahorski et al., 1985; Bylund et al., 1988); yohimbine and rauwolscine are more potent than phentolamine and idazoxan on _2A-adrenoceptors, whereas reversed relative potencies are observed for _2D-adrenoceptors (Starke, 1981; Ennis, 1985; Lattimer and Rhodes, 1985; Alabaster et al., 1986; Limberger et al., 1989). The comparison of the functional potency of several antagonists with their affinity to all subtypes, as determined either in radioligand assays in native tissues possessing only one subtype or in cells transfected with recombinant ₂-adrenoceptors, shows full agreement. So, this functional approach has been extensively used to characterize ₂-autoreceptor subtypes in the different tissues (Hieble et al., 1996). Systematic studies recently undertaken to characterize prejunctional ₂-adrenoceptor subtypes in different species confirmed that receptors with _2A properties occur in some species and receptors with _2D properties occur in others (Bylund et al., 1994; Starke et al., 1995; Trendelenburg et al., 1996; Paiva et al., 1997; Guimarães et al., 1998) (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Distribution of 2-adrenoceptor subtypes in blood vessels

However, some rare discrepancies to this postulate have been reported: in the rat vena cava (Molderings and Göthert, 1993) and rat atria (Connaughton and Docherty, 1990), where the prejunctional receptors were classified as _2B, and in the human kidney cortex (Trendelenburg et al., 1994) and human right atrium (Rump et al., 1995), where they appeared to belong to the _2C-subtype. However, a reinvestigation of these unexpected subclassifications showed that the prejunctional receptors in rat vena cava and atria and in guinea pig urethra were _2D, and those of human kidney were _2A. Thus, in contrast to previous suggestions, all these receptors conform to the rule that ₂-autoreceptors belong, at least predominantly, to the genetic _2A/D-subtype (Trendelenburg et al., 1997).

Although the vast majority of tissues express more than one subtype, there are rare tissues expressing only one subtype: _2A in human platelets (Bylund et al., 1988), _2B in the rat neonatal lung (Bylund et al., 1988), _2C in opossum cells (Murphy and Bylund, 1988), and _2D in the rat submaxillary gland (Michel et al., 1989).

₂-Adrenoceptors are predominantly coupled to the inhibitory heterotrimeric GTP-binding protein inhibiting the activity of adenylyl cyclase (Cotecchia et al., 1990; Wise et al., 1997), inhibiting the opening of voltage-gated Ca²⁺ channels (Cotecchia et al., 1990) and activating K⁺ channels (Surprenant et al., 1992). The ₂-adrenoceptors may also couple to other intracellular pathways involving Na⁺/H⁺ exchange and the activation of phospholipase A₂, C, and D (Limbird, 1988; Cotecchia et al., 1990; MacNulty et al., 1992; Kukkonen et al., 1998). In neurons, ₂-adrenoceptors inhibit N-, P-, and Q-type voltage-gated Ca²⁺ channels (Waterman, 1997; Delmas et al., 1999; Jeong and Ikeda, 2000).

Like the ₁-adrenoceptors, the three ₂-adrenoceptor subtypes are regulated differentially. Human _2C-adrenoceptors do not appear to down-regulate following exposure to agonists (Eason and Liggett, 1992; Kurose and Lefkowitz, 1994); _2A/D- and _2B-adrenoceptors down-regulate apparently due to an increase in the rate of receptor disappearance (Heck and Bylund, 1998).

Three distinct -adrenoceptor subtypes have been cloned so far: ₁, ₂, and ₃ (Bylund et al., 1994). These subtypes are encoded by three different genes located on human chromosomes 10 (₁), 5 (₂), and 8 (₃). The human ₃-adrenoceptor has 49 and 51% overall homology at the amino acid level with human ₂- and ₁-adrenoceptors, respectively (Emorine et al., 1989; Granneman and Lahners, 1994). Other species homologs of the human ₃-adrenoceptor have also been cloned (for a review, see Strosberg, 1997). ₁ and ₂-Adrenoceptors are well known pharmacologically since the classical papers by Lands et al. (1967a,b). They mediate cardiovascular responses to noradrenaline released from sympathomimetic nerve terminals and to circulating adrenaline. They are stimulated or blocked by many compounds that are used to treat important and common diseases, such as hypertension, cardiac arrhythmias, and ischemic heart disease.

The existence of a third -adrenoceptor subtype (₃-adrenoceptor), which was previously shown to mediate lipolysis in rat adipocytes (Harms et al., 1974; Arch et al., 1984; Wilson et al., 1984; Bojanic et al., 1985; Emorine et al., 1989), was also found in blood vessels where it mediates vasodilation (Cohen et al., 1984; Molenaar et al., 1988; Rohrer et al., 1999). ₃-Adrenoceptors are not blocked by propranolol, and other conventional -adrenoceptor antagonists are activated by ₃-adrenoceptor selective agonists like BRL 37344 and CL 316243 (for reviews, see Manara et al., 1995; Strosberg, 1997; Summers et al., 1997; Fischer et al., 1998) and are blocked by ₃-adrenoceptor antagonists like SR-59230, which has been described as ₃-adrenoceptor selective in rat brown adipocytes (Nisoli et al., 1996), rat colonic motility assays (Manara et al., 1996), and human colonic circular smooth muscle relaxation activity assays (De Ponti et al., 1996). More recently, Candelore et al. (1999) did not confirm the selectivity of SR-59230 for human ₃-adrenoceptors, but described two compounds, namely L-748328 and L-748337 that display greater than 90-fold selectivity for human ₃- versus ₁-adrenoceptors, and 20- and 45-fold selectivity versus human ₂-adrenoceptors, respectively. The pharmacology of ₃-adrenoceptors is clearly distinct from that of ₁- and ₂-adrenoceptors; however, one has to bear in mind that there are differences between rodents, where ₃-adrenoceptors were studied initially, and humans, and this contributes to some confusion in the subclassification of -adrenoceptors (Wilson et al., 1996; Arch, 1998). Furthermore, there are also differences depending on the methodological approach used. For example, the potency of catecholamines at the human ₃-adrenoceptor was found to be 1 to 2 orders of magnitude higher when determined in an intact cell cAMP accumulation assay than in a membrane-based adenylyl cyclase activation assay (Wilson et al., 1996).

On the basis of many pharmacological and molecular studies, the existence of a fourth -adrenoceptor subtype was postulated (for reviews, see Arch and Kaumann, 1993; Barnes, 1995; Strosberg and Pietri-Rouxel, 1996; Kaumann, 1997; Strosberg, 1997; Summers et al., 1997; Galitzky et al., 1998; Strosberg et al., 1998; Brodde and Michel, 1999). These receptors would include the receptor in rat soleus muscle, which mediates glucose uptake (Roberts et al., 1993) and the receptor in human and rat heart, which mediates positive chronotropism and inotropism (Kaumann and Molenaar, 1996, 1997; Kaumann et al., 1998; Oostendorp and Kaumann, 2000) (putative ₄-adrenoceptor). A receptor cloned from turkey (_t-adrenoceptor) has no mammalian counterpart (Chen et al., 1994). In mouse brown adipose tissue (±)-CGP-12177, a partial agonist at ₃-adrenoceptors, which is also antagonist at ₁- and ₂-adrenoceptors, evoked a full metabolic response that was of a similar magnitude in wild-type and ₃-adrenoceptor knockout mice; however, the metabolic response to CL-316243 was abolished (Preitner et al., 1998). This unexpected result supports the view that a new -adrenoceptor, distinct from ₁-, ₂-, and ₃-adrenoceptor and referred to as putative ₄-adrenoceptor, is present in brown adipose tissue and can mediate a maximal lipolytic stimulation (Preitner et al., 1998). A similar occurrence was reported for the heart. In ₃-adrenoceptor knockout mice, CGP-12177A increased the force and rate of atrial contractions, and these effects were not antagonized by propranolol, but were antagonized by bupranolol (Kaumann et al., 1998). Furthermore, the binding of ()-[³H]CGP-12177A was similar in ventricular membranes from hearts of wild-type and ₃-adrenoceptor knockout mice; this provides evidence that the cardiac putative ₄-adrenoceptor is distinct from the ₃-adrenoceptor (Kaumann et al., 1998). More recently, evidence was obtained that this putative fourth -adrenoceptor subtype is a particular state of ₁-adrenoceptor (see Section III.C.1.c.).

All -adrenoceptor subtypes signal by coupling to the stimulatory G-protein G_s leading to activation of adenylyl cyclase and accumulation of the second messenger cAMP (Dixon et al., 1986; Frielle et al., 1987; Emorine et al., 1989). However, some recent studies indicate that, under certain circumstances, -adrenoceptors, and particularly the ₃-adrenoceptor, can couple to G_i as well as to G_s (Asano et al., 1984; Chaudry et al., 1994; Xiao et al., 1995; Gauthier et al., 1996).

Intracellular events following -adrenoceptor activation are also linked to ion transport. It is well known, for example, that protein kinase A activated by cAMP phosphorylates L-type Ca²⁺ channels, facilitating Ca²⁺ entry, and producing the positive inotropic effect in atria and ventricles, increased heart rate in the sino-auricular node, and accelerated the conduction in the atrio-ventricular node. In addition to mechanisms that indirectly lead to alterations in ion transport, -adrenoceptor activation is more directly linked to ion channels: -adrenoceptor stimulation is able to activate L-type Ca²⁺ channels via G_s (Brown, 1990); in airway smooth muscle, -adrenoceptor activation opens Ca²⁺-dependent K⁺ channels and charybdotoxina specific inhibitor of the high conductance Ca²⁺-activated K⁺ channelantagonizes the relaxant effects of -adrenoceptor agonists (Miura et al., 1992; Jones et al., 1993).

Multiple mechanisms control the signaling and density of G-protein-coupled receptors. The termination of G-protein-coupled receptor signals involves binding of proteins to the receptor. This process is initiated by serine-threonine phosphorylation of agonist-occupied receptors, both by members of the G-protein-coupled receptor kinase family and by second-messenger-activated protein kinases such as protein kinase A and protein kinase C. Receptor phosphorylation by G-protein-coupled receptor kinase is followed by binding of proteins termed arrestins, which bind to the phosphorylated receptor and sterically inhibit further G-protein activation (Luttrell et al., 1999). Desensitized receptor-arrestin complexes undergo arrestin-dependent targeting for sequestration through clathrin-coated pits (Goodman et al., 1996; Luttrell et al., 1999). Sequestrated receptors are ultimately either dephosphorylated and recycled to the cell surface or targeted for degradation (Luttrell et al., 1999).

In addition, many other G-protein-coupled receptors are sequestrated from the cell membrane and become inaccessible to their ligands. Both receptor/G-protein uncoupling and receptor sequestration may involve the participation of arrestins or other proteins. A model for receptor regulation has been developed on the basis of data from studies of the -adrenoceptors. However, according to recent reports, other G-protein-coupled receptors, like muscarinic receptors in the cardiovascular system, may be regulated by mechanisms other than those that regulate the -adrenoceptors (for a review, see Bünemann et al., 1999).

	III. Postjunctional Adrenoceptors in Vascular Smooth Muscle

Top Previous Next References

Because vascular smooth muscles possess both - and -adrenoceptors, the net response to agonists that like adrenaline stimulate both types of receptors depends on the relative importance of each population. For example, while in the dog saphenous vein, in vitro adrenaline causes contraction, which is enhanced by -adrenoceptor blockade (Guimarães, 1975); in the rabbit facial vein, adrenaline causes relaxation, which is enhanced by -adrenoceptor blockade (Pegram et al., 1976). On the other hand, the contractile response of the saphenous vein to adrenaline is converted into a relaxation when an -adrenoceptor antagonist is present (Guimarães and Paiva, 1981a), and the relaxation caused by adrenaline in the rabbit facial vein is converted into a contraction when a -adrenoceptor antagonist is present (Pegram et al., 1976). Thus, while in the dog saphenous vein, the -adrenoceptor-mediated influence dominates, in the rabbit facial vein the dominating influence is exerted by -adrenoceptor.

In the vast majority of vascular tissues, -adrenoceptor-mediated effects predominate, such that to demonstrate in vitro -adrenoceptor-mediated responses using adrenaline as agonist, both -adrenoceptor blockade and active tone of the tissue must be present. When a pure or almost pure -adrenoceptor agonist like isoprenaline is used, the only requirement to obtain -adrenoceptor-mediated responses is the presence of tone. The threshold for -adrenoceptor-mediated effects in large arteries and veins is between 1 and 10 nM noradrenaline (Guimarães, 1975; Bevan, 1977). The levels of noradrenaline and adrenaline in human arterial plasma at rest are about 2 and 0.5 nM, respectively (Engleman and Portnoy, 1970; DeQuattro and Chan, 1972). In the dog, the level of noradrenaline is similar. Thus, at rest, most vessels are scarcely influenced by circulating catecholamines. However, in the rat mesentery, precapillary sphincters have a threshold response to adrenaline and noradrenaline of 0.1 to 1 nM (Altura, 1971), and rat plasma adrenaline and noradrenaline levels average 2.5 and 3 nM, respectively (Donoso and Barontini, 1986). Although in vivo sensitivity cannot be directly related to plasma catecholamine levels, these data suggest that precapillary sphincters may be affected by circulating catecholamines even under resting conditions, in contrast to other vessels. In humans, during exercise, plasma noradrenaline and adrenaline may reach levels 30 times higher than those at rest, which may have a profound effect on vessels.

It is important to underline that many of the advances made in the last years in the field of receptors in general and on vascular adrenoceptors in particular were due to the possibility to generate knockout mice. However, one should not forget that the lack of a given receptor from conception may be compensated by adequate adjustments, whereas its functional elimination by an antagonist is not acutely compensated (Rohrer and Kobilka, 1998). This is something one must bear in mind when results obtained in wild-type mice are compared with results obtained in knockout mice. It is dangerous to assume that knockout animals differ from the wild-type by no more than the absence of one receptor subtype.

1. In Vitro. In most mammalian species, contraction of vascular smooth muscle is predominantly mediated via ₁-adrenoceptors. Although the existence of both ₁- and ₂-adrenoceptors has been shown by functional studies in vivo, it has been difficult to demonstrate functional postjunctional ₂-adrenoceptors in most arteries in vitro (De Mey and Vanhoutte, 1981; McGrath, 1982; Timmermans and van Zwieten, 1982; Polónia et al., 1985; Guimarães, 1986; Aboud et al., 1993; Burt et al., 1995, 1998). In isolated canine aorta and canine femoral, mesenteric, jejunal, renal, and splenic arteries, contractile responses were exclusively ₁-adrenoceptor-mediated (Polónia et al., 1985; Shi et al., 1989; Daniel et al., 1999). In the arteries of other mammalian species, ₁-adrenoceptors also predominate: in rat aorta (Han et al., 1990; Aboud et al., 1993); in rat carotid, mesenteric, renal, and tail arteries (Han et al., 1990; Villalobos-Molina and Ibarra, 1996); and in human arteries (Flavahan et al., 1987a).

View this table: [in this window] [in a new window]   TABLE 2 Distribution of 1-adrenoceptor subtypes in blood vessels

2. In Vivo. There is also longstanding evidence that multiple ₁-adrenoceptor subtypes are involved in the regulation of peripheral vascular function in vivo (McGrath, 1982; Minneman, 1988; Bylund et al., 1995b). However, the individual contribution of each of the ₁-adrenoceptor subtypes has not been established. Of the three known ₁-adrenoceptor subtypes, _1A- and _1D-adrenoceptors have most often been implicated in the regulation of vascular smooth muscle tone (see Table 2). There are discrepancies between results obtained in vitro and in vivo involving ₁-adrenoceptors. Although in vitro studies in rats had indicated a predominant role of the _1D-adrenoceptor in the vascular contractions caused by ₁-adrenoceptor agonists (Piascik et al., 1995; Hussain and Marshall, 2000), surprisingly experiments in _1B-knockout mice show that the maximal contractile response of aortic rings to phenylephrine was reduced by 40% and the mean arterial blood pressure response to phenylephrine was decreased by 45%, showing that the _1B-adrenoceptor is important for blood pressure and the contractile response of the aorta evoked by ₁-adrenoceptor agonists (Cavalli et al., 1997). In the pithed rat, the systemic blood pressure is tonically regulated by the interaction of peripheral sympathetic nerves with vascular _1A-adrenoceptors (Vargas et al., 1994), although vascular _1D-adrenoceptors have a role in the pressor response to phenylephrine (Zhou and Vargas, 1996). Also, in the pithed rat, it was shown that the selective _1D-adrenoceptor antagonist BMY-7378 not only antagonized the pressor effect of phenylephrine, but also was more potent in young prehypertensive spontaneously hypertensive rats (SHRs) than in young WKY rats. The presence of _1D-adrenoceptors in the resistance vasculature of prehypertensive and hypertensive rats may indicate that they are involved in the development/maintenance of hypertension (Villalobos-Molina et al., 1999). Thus, it may be concluded that, in rats in vivo, the pressor response to phenylephrine is mediated by vascular _1A- and _1D-adrenoceptors (Vargas et al., 1994; Guarino et al., 1996; Zhou and Vargas, 1996).

3. ₁-Adrenoceptor Antagonists in the Symptomatic Treatment of Prostatic Hypertrophy. Clinical interest in this target comes from the fact that selective _1A-adrenoceptor antagonists may have significant therapeutic advantages over nonsubtype selective ₁-adrenoceptor antagonists in the treatment of benign prostatic hypertrophy. Which is the basis for the hypothetical differential effect of _1A-adrenoceptor antagonists at vascular tissue and prostate? Are ₁-adrenoceptors of vascular- and prostatic smooth muscle different? Several studies have shown that the _1A-adrenoceptor subtype accounts for the majority of ₁-adrenoceptor mRNAs and expressed protein in human prostatic smooth muscle and mediates contraction in this tissue (Price et al., 1993; Faure et al., 1994; Lepor et al., 1995; Michel et al., 1996; Schwinn and Kwatra, 1998). However, recent experiments carried out in rat mesenteric arteries (a tissue the ₁-adrenoceptors of which, like those of the prostate, have low affinity for prazosin and RS-17053) (Ford et al., 1996), showed that the affinity of prazosin and RS-17053 was not altered by changing the experimental conditions (lowering temperature, inducing tone via KCl or U-46619a derivative of prostaglandin F₂), calling again our attention to the problem of the putative _1L-adrenoceptors (Yousif et al., 1998; Stam et al., 1999). On the other hand, which is the ₁-adrenoceptor subtype that mediates contractile vascular responses in humans? The few reports on ₁-adrenoceptors in resistance arteries failed to show that a particular ₁-adrenoceptor subtype is of primary importance in the sympathetic control of these vessels. Probably, as animal studies have suggested, each vessel possesses mixtures of ₁-adrenoceptor subtypes, and responses to ₁-adrenoceptor agonists are due to stimulation of more than one subtype (Michel et al., 1998b; Ruffolo and Hieble, 1999; Zhong and Minneman, 1999; Argyle and McGrath, 2000). In a very recent study, it was shown that the receptor subtype mediating the constriction of canine resistance vessels is an _1A-/_1L adrenoceptor (Argyle and McGrath, 2000), which is the same that has been proposed as mediating the adrenergic responses in prostate (McGrath et al., 1996). Thus, the relative selectivity of _1A-adrenoceptor antagonists, if there is any, may not depend on differences between subtypes, but rather on differences between local functional expressions of the receptors. In single human prostatic smooth muscle cells, MacKenzie et al. (2000) showed that the affinity of a prazosin analog for native human _1A-adrenoceptors was higher than for human cloned _1A-adrenoceptors expressed in cell cultures. This suggests that a tissue-specific affinity state of the same receptor genotype exists, and this could be a potential differentiator of drug action (MacKenzie et al., 2000).

B. ₂-Adrenoceptors

1. In Vitro. At the postjunctional level, ₂-adrenoceptors were not found in vitro in the vast majority of the arterial vessels (Table 1). No constrictor activity of ₂-adrenoceptor agonists is present in large arteries; when it appears, it is generally restricted to small arteries/arterioles (Docherty and Starke, 1981; Polónia et al., 1985; Aboud et al., 1993; Leech and Faber, 1996; Daniel et al., 1999). Using rabbit polyclonal antibodies for the ₂-adrenoceptor subtypes, it was observed that _2A/D- and _2C-adrenoceptors are present in the smooth muscle of mouse tail arteries, the expression of _2C-adrenoceptors being smaller in distal arteries than in proximal arteries (Chotani et al., 2000). In contrast to the difficulty in demonstrating postjunctional ₂-adrenoceptors in arteries in vitro, they are consistently found in many isolated veins of different species (De Mey and Vanhoutte, 1981; Constantine et al., 1982; Shoji et al., 1983; Guimarães et al., 1987). This is why the characterization of ₂-adrenoceptor subtypes involved in vascular responses to sympathomimetic agonists is being made in veins (or in vivo). The _2A/D-subtype is the predominant one in almost all the veins until now studied: _2A/D in dog saphenous vein (Hicks et al., 1991; MacLennan et al., 1997); _2A/D in rabbit skeletal muscle venules (although predominantly _1D) (Leech and Faber, 1996); and _2A/D (most probably) in the porcine palmar lateral vein (Blaylock and Wilson, 1995). In good agreement with the premise that the _2A- and _2D-adrenoceptors represent species orthologs (Bylund et al., 1995a)_2A occurring in humans, dogs, pigs, and rabbits and _2D occurring in rats, mice, and cowsit was observed that postjunctional ₂-adrenoceptors of the canine mesenteric vein are predominantly _2A, whereas those of the rat femoral vein are predominantly _2D (Paiva et al., 1999). In human saphenous vein, correlation of ₂-adrenoceptors antagonist potency with binding affinity suggests the contribution of the _2C-subtype (Gavin et al., 1997).

2. In Vivo. ₂-Adrenoceptors are essential components of the neural complex system regulating cardiovascular function (Ruffolo et al., 1991) (see Section IV.). When clonidine-like ₂-adrenoceptor agonists are intra-arterially administered to wild-type mice, they cause an initial brief pressor effect that is gradually reversed to hypotension at the same time as the animal experiences a severe bradycardia (Link et al., 1996; MacMillan et al., 1996). This is a typical cardiovascul