I. Introduction

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"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

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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

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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 cardiovascular response to intravenous administration of an ₂-adrenoceptor agonist also in humans and other species (Hoefke and Kobinger, 1966; Kallio et al., 1989; Bloor et al., 1992). Three main factors are now known to participate in this biphasic response: activation of ₂-adrenoceptors on vascular smooth muscle cells that is responsible for the initial and transient hypertensive phase; activation of ₂-adrenoceptors in the brainstem leading to a reduction in sympathetic tone with a resultant decrease in blood pressure and heart rate (this hypotensive effect has been the rationale for the use of clonidine in the treatment of hypertension); and a third factor, namely the stimulation of prejunctional ₂-adrenoceptors located on sympathetic terminals innervating the vascular smooth muscle cells, an effect that augments the hypotensive effect due to stimulation of central ₂-adrenoceptors (Ruffolo et al., 1993; Urban et al., 1995). Bearing in mind that all the three subtypes of ₂-adrenoceptors (_2A/D, _2B, and _2C) are present in the vascular tree (Hicks et al., 1991; Gavin et al., 1997; MacLennan et al., 1997; Paiva et al., 1999), which is the involvement of each subtype, if any, in these responses?

3. Blood Pressure Regulation in ₂-Adrenoceptor-Deficient Mice. Five mouse strains with genetic alterations of ₂-adrenoceptor expression have been generated; they offer new pathways to further identify the role of each subtype (Hein, 1999; Kable et al., 2000): deletion of _2A/D- (_2A/D-knockout), _2B- (_2B-knockout), or _2C-gene (_2C-knockout) (Link et al., 1995, 1996; Altman et al., 1999). More recently, mouse strains lacking _2A/D-, _2B-, or _2C-subtypes were crossed to generate double knockout mice. However, the only viable animals were those lacking both _2A/D- and _2C-adrenoceptors (_2A/DC-knockout) (Hein et al., 1999). Mice have been also developed with a point mutation of the _2A/D-gene (_2A/D-D79N). The D79N mutation causes a replacement of the aspartate with an asparagine residue at position 79, in the second transmembrane domain of the _2A/D-adrenoceptor. In cultured cell lines, the _2A/D-D79N mutant receptor failed to activate K⁺ currents, but exhibited normal inhibition of voltage-gated calcium channels and cAMP production (Surprenant et al., 1992). It was expected that the expression of this mutation in the intact animal would provide insight into the signal transduction mechanisms mediating the effect of _2A-adrenoceptor stimulation. However, _2A/D-D79N mice showed about an 80% reduction in ₂-adrenoceptor binding, as determined by radioligand studies in the brain (MacMillan et al., 1996). Thus, the _2A/D-D79N receptor expressed in vivo showed different characteristics, compared with its expression in vitro, thus behaving as a functional knockout.

C. -Adrenoceptors

1. In Vitro a. ₁- and ₂-Adrenoceptors. According to Lands and coworkers (1967a,b), the -adrenoceptors in the peripheral vessels were classified as ₂. Later studies using more selective agonists and antagonists showed that relaxation of vascular smooth muscle cells resulted from activation of either ₁- or ₂-adrenoceptor subtypes, and that the involvement of each subtype depended on the vascular bed and the species (O'Donnell and Wanstall, 1984; Guimarães et al., 1993; Shen et al., 1994, 1996; Begonha et al., 1995). ₂-Adrenoceptors represent the predominant subtype in most vascular smooth muscles, although ₁-adrenoceptors may contribute to vasodilation (for a review, see Osswald and Guimarães, 1983). In a few vessels, ₁-adrenoceptors appear to predominate, e.g., coronary arteries (O'Donnell and Wanstall, 1985; Begonha et al., 1995) and cerebral arteries (Edvinsson and Owman, 1974). In contrast to the heart where a maximal increase in force of contraction is obtained by stimulation of ₁-adrenoceptors only and activation of ₂-adrenoceptors causes no more than a submaximal effect (Kaumann et al., 1989; Motomura et al., 1990), in the vessels (at least in the veins), the maximum relaxation evoked by ₂-adrenoceptors is larger than that evoked by ₁-adrenoceptor stimulation (Guimarães and Paiva, 1981c) (Fig. 1). The maximal -adrenoceptor-mediated relaxation varies from vascular bed to vascular bed (see Section VI.C.) and crucially depends on the level of the tone of the tissue (Guimarães, 1975; Begonha et al., 1995) (Table 3). The maximal -adrenoceptor-mediated relaxation of the canine veins vary markedly from vein to vein (Table 4). After precontraction with methoxamine, isoprenaline antagonized more than 80% of the precontraction in cephalic, external jugular, azygos, renal, femoral, saphenous, pulmonary, splenic, and the superior part of the inferior vena cava; whereas in the portal, mesenteric, and inferior segments of vena cava, the maximal relaxation antagonized less than 25% of the previous contraction (Furuta et al., 1986).

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Dog saphenous vein strips. After contractions of about the same magnitude (75% of the maximum) caused by noradrenaline (300 nM) (NA) or adrenaline (510 nM) (AD), concentration-response curves (relaxation) to terbutaline (Terb) or dobutamine (Dob) were determined. Because the concentration of noradrenaline used to cause the precontraction also occupies 1-adrenoceptors, the relaxation to the selective 1-adrenoceptor agonist dobutamine was much smaller than that to terbutaline. Conversely, because the concentration of adrenaline used to precontract the tissue also occupies 2-adrenoceptors, relaxation to the selective 2-adrenoceptor agonist terbutaline was much smaller than that to dobutamine. Furthermore, the maximum relaxation to terbutaline was greater than that to dobutamine (Guimarães and Paiva, 1981c).

b. ₃-Adrenoceptors. The participation of a third -adrenoceptor subtype in -adrenoceptor-mediated vasodilatation was suggested by results obtained in several studies. Pindolol, a nonselective -adrenoceptor antagonist with significant agonist activity, caused relaxation of canine isolated perfused mesenteric vessels (Clark and Bertholet, 1983

c. Putative ₄-Adrenoceptors. Although there is now convincing evidence supporting the functional presence of ₃-adrenoceptors in vascular tissue, various observations cannot be fully explained by the existence of ₁-, ₂-, and ₃-adrenoceptors only. For example, the propranolol-resistant component of the effect of isoprenaline in rat aorta is not antagonized by either the selective ₃-adrenoceptor antagonist SR-59230 (Brawley et al., 2000a

2. In Vivo. -Adrenoceptor-mediated vasodilation is thought to play an important physiological role in the regulation of vascular tone. Stimulation of peripheral -adrenoceptors leads to relaxation of the vascular smooth muscle, thereby controlling the peripheral vascular resistance and consequently the distribution of blood to the different organs. During exercise, for example, activation of -adrenoceptors contributes to the increased blood flow to skeletal muscle.

Although much has been learned about the role of the individual -adrenoceptor subtype using classical pharmacological approaches in vitro, experiments in awake and unrestrained animals are of crucial importance to determine the real influence of factors coming into play, since reflex pathways can be obscured in anesthetized animals and are absent in the in vitro preparations. Experiments carried out in vivo in wild-type animals of several species had already indicated that the activation of ₂- and ₁-adrenoceptors led to relaxation of vascular smooth muscle of both arteries and veins (Taira et al., 1977

View larger version (16K): [in this window] [in a new window]   Fig. 2.   The effects of CL-316243 on mean arterial pressure and total peripheral resistance are compared in the absence (open bars) and presence (closed bars) of 1-/2-adrenoceptor blockade. Graded decrease in responsiveness to CL-316234 can be seen from the dog to the rat and to the monkey. 1-/2-Adrenoceptor blockade did not affect significantly either parameter. Adapted from Shen et al. (1996).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Hemodynamic responsiveness to the 3-adrenoceptor agonist CL-316243 (CL; open bars) and the nonselective -adrenoceptor agonist isoprenaline (Iso; closed bars). Upper panel shows a blood pressure tracing of wild-type mouse given a single bolus injection of CL-316243 (100 µg/kg). Ten minutes later, isoprenaline (3 µg/kg i.a.) was administered. Bar graph in the lower panel shows the effects caused by the same drugs under identical conditions in 1-, 2-, and 1-/2-knockout (KO) animals. Adapted from Rohrer et al. (1999).

	IV. Prejunctional Adrenoceptors

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At the prejunctional level, ₂-adrenoceptors have been found in vitro in every vascular tissue (arteries and veins) until now studied where they mediate a negative modulation of the release of noradrenaline (Starke, 1987; Langer, 1997). Experiments in isolated organs support the view that the _2A/D-subtype is the principal prejunctional ₂-adrenoceptor; some studies indicate that, in certain tissues, including the heart and some vessels, another ₂-subtype might also be involved in the regulation of the release of noradrenaline (Oriowo et al., 1991; Limberger et al., 1992; Guimarães et al., 1997; Trendelenburg et al., 1997; Docherty, 1998; Ho et al., 1998). Results obtained in knockout mice clearly confirmed this hypothesis. In the vas deferens, for example, it was observed that maximal inhibition by ₂-adrenoceptor agonists of the electrically evoked contractions was reduced to 50% in mice lacking _2A/D, whereas the effect of these agonists in _2C-adrenoceptor-deficient mice was unchanged. Furthermore, in mice lacking both the _2A/D- and the _2C-adrenoceptors, the prejunctional effect of ₂-adrenoceptor agonists was abolished, indicating that the residual 50% response to ₂-adrenoceptor agonists in _2A/D-knockout animals was due to prejunctional _2C-adrenoceptors (Hein et al., 1998). Also in the heart atria and in the brain cortex, deletion of _2A/D-adrenoceptors reduced, but did not abolish, the inhibitory effect of the ₂-selective agonist UK-14304, indicating that a second ₂-autoreceptor operates in both sympathetic and central adrenergic neurons. However, the loss in E_max of ₂-adrenoceptor agonists was smaller in the heart than in the brain, supporting the view that, in the peripheral tissues, prejunctional non-_2A/D-adrenoceptors are functionally more important (Hein et al., 1999; Trendelenburg et al., 1999). Again, in _2A/DC-knockout mice, the concentration-dependent inhibition of noradrenaline release caused by ₂-adrenoceptor agonists in the atria was abolished, indicating that the _2C-adrenoceptor is the second type involved in regulating noradrenaline release (Hein et al., 1999). Thus, the hypothesis that more than one ₂-adrenoceptor subtype might be present at the prejunctional level in the same tissue was confirmed in knockout animals.

According to Link et al. (1992), noradrenaline has a higher affinity for _2C- than for _2A/D-adrenoceptors. In mouse atria, Hein et al. (1999) confirmed that noradrenaline was more potent on _2C- than on _2A/D-adrenoceptors (EC₅₀ = 16 nM, 20 nM, and 156 nM in wild-type, _2A/D-knockout, and _2C-knockout animals, respectively) and showed that _2C-adrenoceptors inhibit transmitter release at low levels of sympathetic tone and that _2A/D-adrenoceptors are required to control release at higher levels of sympathetic tone.

Unfortunately there are no studies in isolated vascular tissues from knockout animals. However, the calculation of the correlation between the pK_i values of several antagonists at some canine and human vessels and their pK_d values at prototypical _2A, _2B, _2C, and _2D radioligand binding sites (Altman et al., 1999) shows that, as in other peripheral tissues, the main modulatory role is played by _2A/D-adrenoceptors and suggests that more than one ₂-adrenoceptor subtype participate in the feedback inhibition of transmitter release (Table 5). This calculation still suggests that _2B or _2C or both may be involved in this modulatory influence. Additionally, it can be concluded that the existence of a second type of prejunctional receptor in vascular tissues of _2A/D-knockout mice does not result from a compensatory mechanism (Altman et al., 1999), since these results were obtained in wild-type animals.

There is no doubt that in vessels the control mechanism of noradrenaline release is highly active at physiological frequencies of electrical stimulation. Evidence supporting the possibility of a tonic physiological role of a negative feedback control of noradrenaline release appeared before the discovery of prejunctional -adrenoceptors. The first report pointing in this direction was that by Malik and Muscholl (1969), who showed that noradrenaline, in doses that did not alter basal resistance, slightly reduced the response of the perfused mesenteric artery to sympathetic nerve stimulation. This was confirmed by the observation, in the rabbit pulmonary artery, that the inhibitory influence of a fixed concentration of an exogenous agonist was not as effective at high as at low frequencies of stimulation (Starke and Endo, 1975). Indeed, at the higher frequencies, the negative influence of -adrenoceptor agonists is less because the prejunctional -adrenoceptors are already more intensely activated by endogenous noradrenaline (Vizi et al., 1973; McCulloch et al., 1975).

Numerous pharmacological findings obtained by many authors in several species, including humans, support the view that a negative feedback mechanism mediated by prejunctional ₂-adrenoceptor operates under physiological conditions of noradrenergic neurotransmission (for reviews, see Starke, 1977, 1987; Langer, 1981, 1997). However, this hypothesis was contested by Kalsner (1982) and Kalsner and Westfall (1990). The latest observations in knockout animals clearly show that prejunctional ₂-adrenoceptors are really autoreceptors that accomplish a physiological function.

The release of noradrenaline from sympathetic varicosities is modulated by a large number of prejunctional auto- and heteroreceptors. Facilitatory -adrenoceptors have been shown in various sympathetically innervated tissues of many species both in vitro (Adler-Graschinsky and Langer, 1975; Majewski, 1983; Misu and Kubo, 1986; Encabo et al., 1996) and in vivo (Boudreau et al., 1993; Tarizzo et al., 1994; Vila and Badia, 1995). Those receptors, which have been demonstrated to be of the ₂-subtype (Dahlöf et al., 1978; Guimarães et al., 1978; Göthert and Hentrich, 1985; Coopes et al., 1993), may be activated by circulating adrenaline (Stjärne and Brundin, 1975) and/or by adrenaline taken up by and released from sympathetic varicosities as a cotransmitter (Majewski et al., 1981a,b; Misu et al., 1989; Tarizzo and Dahlöf, 1989). Very recently, the existence of prejunctional ₂-adrenoceptors located on nerve terminals that release nitric oxide (NO: NOergic nerve terminals) of the porcine basilar arteries was proposed by Lee et al. (2000). According to these authors, noradrenaline released from sympathetic nerves upon application of nicotine, acts on prejunctional ₂-adrenoceptors of NOergic nerve terminals to release NO, resulting in vasodilatation. In humans, there is also evidence for facilitatory -adrenoceptors on noradrenergic nerve endings, both in vitro (Stjärne and Brundin, 1975; Stevens et al., 1982) and in vivo (Brown and Macquin, 1981; Vincent et al., 1982; Blankestijn et al., 1988). However, whereas prejunctional ₂-adrenoceptors (of the _2A/D- and _2C-subtype) play an important physiological role in the feedback inhibition of neurotransmitter release (Starke, 1987; MacMillan et al., 1996; Altman et al., 1999; Trendelenburg et al., 1999), the physiological role of the facilitatory prejunctional -adrenoceptors remains controversial (Abrahamsen and Nedergaard, 1989, 1990, 1991; Floras, 1992; Apparsundaram and Eikenburg, 1995; Coopes et al., 1995). A hypothetical pathological implication of the facilitatory prejunctional ₂-adrenoceptors originated when Guimarães et al. (1978) first showed that, in canine saphenous vein, propranolol was able to reduce the electrically evoked tritium overflow after preloading of the venous tissue with ³H-adrenaline, but not when it had been preloaded with ³H-noradrenaline. Confirmatory results were obtained in different in vitro vascular peparations (Majewski et al., 1981a; Misu et al., 1989; Rump et al., 1992) and also in vivo studies: in the anesthetized rabbit (Majewski et al., 1982); in the anesthetized dog (Boudreau et al., 1993); in the pithed rat (Vila and Badia, 1995); and in the demedulated rat (Coopes et al., 1993, 1995). On the basis of these results, the hypothesis was put forward that high levels of adrenaline within the synaptic cleft could either directly stimulate prejunctional ₂-adrenoceptors or be taken up by sympathetic nerves, and then be coreleased and activate prejunctional ₂-adrenoceptors. In both cases, stimulation of this positive feedback loop would lead to an increased release of noradrenaline, which might represent an early step in the development of hypertension (Majewski et al., 1981b; Misu et al., 1990; for a review, see Floras, 1992). Depletion of plasma adrenaline by surgical adrenal demedullation attenuates the development of hypertension in 4-week-old SHRs (Borkowski and Quinn, 1985), and this effect is antagonized by depot implants of adrenaline. Moreover, the prohypertensive effect of adrenaline in demedullated SHRs was abolished by concomitant treatment with ₂-adrenoceptor antagonists (Borkowski and Quinn, 1985). The development of hypertension was attenuated only in SHRs demedullated at 6 weeks of age or younger, indicating that a period of critical sensitivity of prejunctional ₂-adrenoceptors to the facilitatory effect of catecholamines may exist (Borkowski, 1991). In humans, episodes of sympathoadrenal activation repeatedly causing increases in plasma adrenaline concentration might, by direct (as a hormone), indirect (as a cotransmitter), or both ways, initiate or facilitate the development of primary hypertension (Brown and Macquin, 1981; Blankestijn et al., 1988). Although attractive and based on a large amount of experimental work, this theory has not received conclusive support so far. Floras et al. (1988) reported that 30 min after a local infusion of adrenaline into the forearm of volunteers, there was a facilitation of neurogenic vasoconstriction that was due to a delayed facilitation of noradrenaline release caused by the previous infusion of adrenaline. Stein et al. (1997), under identical conditions, did not observe any delayed facilitatory effect on noradrenaline spillover in the forearm of normotensive or borderline hypertensive subjects. However, these authors observed an increase in systemic noradrenaline spillover 30 min after the infusion of adrenaline, suggesting that there is a delayed facilitatory response to adrenaline in specific organs, the identification of which would be of importance in elucidating the role of this mechanism in the pathogenesis of hypertension. In a comparative study involving patients with longstanding essential hypertension and normotensive control subjects, Chang et al. (1994) evaluated the kinetics of noradrenaline by measuring in the forearm the appearance rate of noradrenaline in plasma and the spillover of noradrenaline into plasma before and after the infusion of adrenaline and found no differences between the two groups. Similarly, in 40 healthy volunteers, loading of sympathetic nerve terminals of the human forearm with adrenaline did not augment subsequent neurogenic vasoconstriction or noradrenaline release in response to sympathetic stimulation (Goldstein et al., 1999). Using a different methodological approach, Thompson et al. (1998) also obtained negative results: isometric handgrip contraction evoked similar responses in total and cardiac noradrenaline spillovers, and in muscle sympathetic activity before and after an infusion of adrenaline. The influence of muscle contraction and blood flow on noradrenaline and adrenaline spillover was also studied in the in situ canine gracilis muscle, and spillover of both amines was observed (Lavoie et al., 2000). Since adrenaline is not synthesized locally, adrenaline spillover means that it was taken up from the circulation, stored in the vesicles, and then re-released with noradrenaline (Lavoie et al., 2000).

Although convincing evidence for the positive feedback loop hypothesis is still lacking, it may be inadvisable to regard it as disproven. As suggested by Folkow (1982), the development of hypertension by this mechanism may be restricted to a specific subset of individuals genetically predisposed to high blood pressure. As pointed out by Floras (1992), there are no prospective evaluations of the predictive value of augmented plasma adrenaline concentrations in childhood and adolescence as an indicator for later hypertension development; it is possible that, as shown for SHRs (Borkowski 1991), a period of critical sensitivity of prejunctional ₂-adrenoceptors to catecholamines exists during which the pathological development starts. If this were true for humans and if it were known into which age this critical period fell, an adequate treatment might avoid the progress toward an established hypertension.

	V. Endothelial Adrenoceptors

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It is now widely accepted that vascular endothelium plays an important role in the function of the cardiovascular system (Moncada et al., 1991a; Vaz-da-Silva et al., 1996; Busse et al., 1998). Functional evidence suggesting that ₂-adrenoceptors play a role in the physiology of the vasculature was first reported by Cocks and Angus (1983), who showed that, in isolated coronary, renal, and mesenteric arteries, noradrenaline and clonidine caused relaxation that was inhibited by selective ₂-adrenoceptor antagonists and was eliminated by removal of the endothelium. Similar results were obtained in several isolated arteries (Egleme et al., 1984; Angus et al., 1986) and veins (Miller and Vanhoutte, 1985). Soon after the discovery of the EDRF (Furchgott and Zawadzki, 1980)later on identified with NO(Ignarro et al., 1987; Palmer et al., 1987), it was demonstrated that activation of ₂-adrenoceptors on endothelial cells stimulates the release of NO, an action that would tend to attenuate vasoconstriction produced by activation of postjunctional vascular ₁-adrenoceptors (Angus et al., 1986; Vanhoutte and Miller, 1989; Richard et al., 1990). Furthermore, it was suggested that endothelial ₂-adrenoceptors mediate release of EDRF in coronary microvessels (Angus et al., 1986). Thus, it appears that ₂-adrenoceptor agonists do indeed have the capability of modulating vascular responsiveness via stimulation of the release of NO in both large arteries and microcirculation. Furthermore, it was reported that noradrenaline-induced release of nitric oxide is enhanced in mineralocorticoid hypertension (Bockman et al., 1992), indicating that endothelial ₂-adrenoceptors may play an important role in the regulation of vascular tone not only in physiological, but also in pathological conditions.

Which ₂-adrenoceptor subtype is responsible for this modulatory influence? The first study aiming at characterizing the ₂-adrenoceptor subtypes present on vascular endothelium was carried out in pig coronary arteries and showed that the endothelium of this vessel possesses both _2A/D- and _2C-adrenoceptors, the latter predominating (77% of _2C versus 23% of _2A/D). However, despite the prominent presence of _2C-adrenoceptors, the _2A/D-adrenoceptor subtype is the one mediating endothelium-dependent relaxation (Bockman et al., 1993). Interestingly, it was shown that in the rat mesenteric artery the ₂-adrenoceptor that is coupled to endothelium-dependent NO-mediated relaxation belongs to the _2A/D-subtype appearing in its _2D-version (Bockman et al., 1996). Also in the endothelium of different species, the _2A/D-adrenoceptors serve the same function (Bylund et al., 1995a). Contrary to what was expected, cAMP is not involved in the signal transduction pathway for _2A/D-adrenoceptor-mediated NO formation (Bockman et al., 1996).

It is now widely accepted that -adrenoceptors exist on endothelial cells and contribute to the regulation of vasomotor tone. The role these receptors play, the mechanisms by which this role is played and the -adrenoceptor subtypes that are involved are still debatable questions. Some of the first studies on the existence of endothelial -adrenoceptors and some others carried out later on did not find evidence supporting their existence. Removal of the endothelium or inhibitors of NO synthase were found to have no influence on isoprenaline-evoked relaxations in rat aorta (Konishi and Su, 1983), canine coronary arteries (Cohen et al., 1983, 1984; White et al., 1986), rat carotid artery (Oriowo, 1994), or human internal mammary artery (Molenaar et al., 1988). In contrast, many other authors reported that removal of endothelium reduces the relaxations caused by -adrenoceptor agonists in several isolated vessels from different species, including humans (Grace et al., 1988; Kamata et al., 1989; Dainty et al., 1990; Gray and Marshall, 1992; Delpy et al., 1996; Toyoshima et al., 1998; Ferro et al., 1999; Trochu et al., 1999; Brawley et al., 2000a,b; Vanhoutte, 2000). Surprisingly, in the same preparation (the thoracic aorta), different authors found opposite results. This discrepancy may be ascribed to one or more of the following factors: 1) the agent used to precontract the vessel was either noradrenaline (which also activates -adrenoceptors) or phenylephrine (which lacks affinity for -adrenoceptors) (Guimarães, 1975); 2) the level of the precontraction at which the -adrenoceptor-mediated relaxation might not be the same and the magnitude of the relaxant effect is critically dependent on the extent of the pre-existing tone (Guimarães, 1975); and 3) the ₃-adrenoceptor agonist used may be more or less active on ₁- and ₂-adrenoceptors.

According to Eckly et al. (1994), the reduction in response to isoprenaline after pretreatment with L-NAME or endothelial removal can be explained by the fact that the precontraction of the vessel is greater than in control tissues due to the disappearance of NO production under resting conditions. This enhancement of the precontraction would counteract the relaxation (Guimarães, 1975). However, it has been shown that endothelial removal does not consistently increase the preconstriction caused by noradrenaline or phenylephrine (Delpy et al., 1996; Brawley et al., 2000b). Many other kinds of evidence also support the view that -adrenoceptors are present in endothelial cells and mediate relaxing responses in which NO is involved. First of all, the presence of -adrenoceptors was confirmed by radioligand binding studies in cultured bovine aortic endothelial cells (Steinberg et al., 1984), by autoradiography in human cardiac endocardium (Buxton et al., 1987), in the endothelium of internal mammary artery and saphenous vein (Molenaar et al., 1988), and by biochemical data obtained in cultured human umbilical vein endothelial cells (Ferro et al., 1999). Furthermore, in vivo studies in cat hindlimb (Gardiner et al., 1991), canine coronary artery (Parent et al., 1993), and newborn pig pial arteries (Rebich et al., 1995) support a role of vascular endothelium in -adrenoceptor-mediated relaxation. In humans, it was also found that forearm blood flow increases by infusion of either isoprenaline or salbutamol into the brachial artery, and coinfusion of the nitric oxide synthase inhibitor L-NMMA blocks this response to either drug (Dawes et al., 1997). Additionally, it had been shown that relaxant responses of the rat aorta to isoprenaline are inhibited by methylene blue and hemoglobin (Grace et al., 1988), indicating that the endothelium-dependent NO/cGMP system may be activated by stimulation of -adrenoceptors (Grace et al., 1988; Gray and Marshall, 1992; Iranami et al., 1996).

The second point concerns the role played by endothelial -adrenoceptors and the mechanism through which they induce their effects. Pretreatment with L-NAME and endothelium removal exert a similar inhibitory influence on isoprenaline-evoked relaxation, and the combination of the two procedures has no additional effect, compared with either treatment alone (Gray and Marshall, 1992; Ferro et al., 1999). On the basis of this evidence, these authors concluded that -adrenoceptor-mediated vasorelaxation is totally endothelium-dependent: isoprenaline-evoked relaxation is due to the elevation of cyclic AMP caused by ₂-adrenoceptor stimulation, and this elevation activates the L-arginine/NO system and gives rise to vasorelaxation (via cGMP formation) (Gray and Marshall, 1992; Ferro et al., 1999). However, other authors found little or no effect of endothelium removal on isoprenaline-evoked relaxations (Konishi and Su, 1983; Moncada et al., 1991b; Eckly et al., 1994; Satake et al., 1996). The hypothesis that incomplete removal of endothelium might account for some remaining relaxation to isoprenaline (Gray and Marshall, 1992) can be discarded at least in some cases in which part of the relaxation to isoprenaline remained, although removal of endothelium had abolished acetylcholine-evoked relaxation (Brawley et al., 2000b). In these cases, treatment with L-NAME of the endothelium-free preparations caused no further effect on the isoprenaline-evoked relaxations (Brawley et al., 2000b). Thus, it appears that the relaxant effect of isoprenaline involves two components: one endothelium-dependent and another endothelium-independent (Brawley et al., 2000b). The endothelium-dependent component is triggered by -adrenoceptor activation and leads to the promotion of NO production/release (Gray and Marshall, 1992; Ferro et al., 1999; Brawley et al., 2000b), or to some kind of enhancement of smooth muscle -adrenoceptor-mediated relaxation by basal release of NO (Grace et al., 1988; Delpy et al., 1996).

A third point refers to the endothelial -adrenoceptor subtype(s) involved in isoprenaline-evoked relaxation of the vascular smooth muscle. It is now recognized that -adrenoceptors located in the endothelium play an important role in the relaxant response to isoprenaline, since the nonselective ₁-and ₂-adrenoceptor antagonist propranolol antagonized this relaxant effect (Oriowo, 1995; Sooch and Marshall, 1996; Brawley et al., 2000a,b). However, recent studies carried out in humanseither in umbilical veins in vitro (Ferro et al., 1999) or in the forearm in vivo (Dawes et al., 1997)showed that vasorelaxation to isoprenaline was abolished by the selective ₂-adrenoceptor antagonist ICI-118551 and remained unchanged in the presence of the ₁-adrenoceptor antagonist CGP-20712, indicating that as in the vascular smooth muscle cells (Lands et al., 1967a,b), the endothelial -adrenoceptors are totally or at least predominantly of the ₂-subtype (Dawes et al., 1997; Ferro et al., 1999). Furthermore, it was observed that, after L-NAME treatment or removal of endothelium, relaxant responses to isoprenaline were still unaffected by propranolol, suggesting that they were mediated by ₃- and/or the low-affinity state of ₁-adrenoceptors, formerly proposed as putative ₄-adrenoceptors (Brawley et al., 1998).

Additionally, it was shown that relaxation of rat thoracic aorta was also caused by selective ₃-adrenoceptor agonists like CGP-12177 (Mohell and Dicker, 1989), cyanopindolol (Engel et al., 1981), ZD-2079 (Grant et al., 1994), ZM-215001 (Tesfamariam and Allen, 1994), and SR-58611 (Trochu et al., 1999), further supporting the presence of ₃-adrenoceptors (Brawley et al., 2000a,b).

Endothelial removal or pretreatment with L-NAME significantly reduced the relaxation caused by isoprenaline or SR-58611 (Trochu et al., 1999), but had less effect on the relaxation caused by another selective ₃-adrenoceptor agonist BRL-37344 (MacDonald et al., 1999) or CGP-12177 (Brawley et al., 2000a,b). Furthermore, sodium nitroprusside enhanced isoprenaline effects as previously reported (Maurice and Haslam, 1990), but had little or no effect on the response to CGP-12177 (Brawley et al., 2000a,b) or on the relaxation to isoprenaline in the presence of propranolol. After L-NAME had reduced responses to both isoprenaline and CGP-12177, sodium nitroprusside restored them, but the contribution of NO to the atypical -adrenoceptor-mediated response was less than to ₁- and ₂-adrenoceptor-mediated relaxation (Brawley et al., 2000a,b). Both of these procedures indicate that exogenously applied NO interacts with the ₁- and ₂-adrenoceptor signaling pathway to a greater extent than with the non-₁-/₂-adrenoceptor pathway. However, the non-₁-/₂-adrenoceptor-mediated component of the response to isoprenaline appears to be partially endothelium-dependent, since L-NAME or endothelium removal attenuated isoprenaline relaxation in the presence of propranolol (Shafiei and Mahmoudian, 1999; Trochu et al., 1999; Brawley et al., 2000b).

All of these findings show that the endothelium/NO pathway modulates ₁- and ₂-adrenoceptor-mediated responses in rat aorta to a greater extent than non-₁-/₂-adrenoceptor-mediated responses (Brawley et al., 1998; MacDonald et al., 1999) and indicate that non-₁-/₂-adrenoceptors are present in the endothelium of some mammalian arteries.

Which non-₁-/₂-adrenoceptor? This is still a difficult question to answer. First of all, in the rat pulmonary vessels, several ₃-adrenoceptor agonists (SR-58611, SR-59119, and SR-59104) caused relaxant effects. However, only the effect of SR-59104 was antagonized by the selective ₃-adrenoceptor antagonist SR-59230 (Dumas et al., 1998). In the rat thoracic aorta, ₃-adrenoceptors are mainly located on endothelial cells, and act in conjunction with ₁- and ₂-adrenoceptors to mediate relaxation through activation of an NO synthase pathway and subsequent increase in cyclic GMP levels (Trochu et al., 1999).

	VI. Distribution of Vascular Adrenoceptors

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In vascular tissue, - and -adrenoceptors are not situated close to each other (Guimarães et al., 1981a,b; Guimarães, 1982), such that when there is a change in the concentration of circulating adrenaline coming from the blood or of noradrenaline coming from the sympathetic nerve terminals, it does not affect - and -adrenoceptors equally; there are two different biophases for sympathomimetic agonists: one for -adrenoceptors around the nerve terminals, where the concentration of the agonist available for -effect is mainly governed by uptake into these terminals; and one for -adrenoceptors in the neighborhood of catechol-O-methyl transferase whose activity is the main factor determining the concentration of the agonist available for the -effect (for a review, see Guimarães, 1982; Guimarães et al., 1982). This will contribute to the fact that the different vessels have different sensitivities to sympathomimetic amines, such that some of them may be under the control of circulating catecholamines, whereas others are not.

In 1980, Yamaguchi and Kopin showed that, in the pithed rat, the pressor response to sympathetic nerve stimulation is the result of activation of ₁-adrenoceptors, whereas the pressor effects of the exogenous catecholamines are medited by ₂-adrenoceptors. This conclusion was consistent with the suggestion that postjunctional vascular ₂-adrenoceptors might be located at extrajunctional sites (Langer et al., 1981). This differential location of ₁- and ₂-adrenoceptors in relation to the nerve terminals was confirmed in conscious rabbits, where it was shown that pretreatment with 6-hydroxydopamine augmented the pressor response to ₁-adrenoceptor agonists without changing the responses to ₂-adrenoceptor agonists (Hamilton and Reid, 1981). All these results are consistent with the hypothesis that ₁-adrenoceptors are located in the vicinity of sympathetic nerve terminals, strategically situated to be activated by noradrenaline coming out from the nerves, whereas ₂-adrenoceptors are situated extrajunctionally and may be activated preferentially by circulating catecholamines, particularly adrenaline. However, in the isolated rabbit portal vein, the selective ₁-adrenoceptor antagonist prazosin failed to antagonize the contractions evoked by electrical stimulation of the vessel, whereas the selective ₂-adrenoceptor antagonist rauwolscine did antagonize these contractions (Docherty and Starke, 1981). In agreement with this report, it was shown that, in the canine saphenous vein, the inhibition of neuronal uptake by cocaine enhanced the contractile response to noradrenaline more in the presence of prazosin (₂-adrenoceptor-mediated response) than in that of yohimbine (₁-adrenoceptor-mediated response) (10- versus 6-fold; Guimarães et al., 1983). Furthermore, yohimbine was more potent than prazosin in antagonizing the effect of noradrenaline released from nerve terminals either by electrical stimulation or tyramine (Guimarães et al., 1983; Cooke et al., 1984; Flavahan et al., 1984; Pereira et al., 1991). More recently, it was reported that, in the canine saphenous vein in vitro, the contractile response evoked by electrical stimulation is mediated by three receptors: ₁- and ₂-adrenoceptors and P2X-receptors; the ₁-adrenoceptor and the P2X-receptor-mediated contractions develop immediately after starting the stimulation and reach the maximum very quickly, whereas the ₂-adrenoceptor-mediated contraction develops slowly, although reaching a maximum of similar magnitude. The purinergic component was smaller than the other two. Cocaine, which did not change the purinergic response, enhanced both adrenoceptor-mediated components but enhanced more markedly the ₂-adrenoceptor-mediated- than the ₁-adrenoceptor-mediated component (Hiraoka et al., 2000).

In surgically denervated canine saphenous veins, Flavahan et al. (1987b) showed that ₂-adrenoceptor-mediated responses to noradrenaline are augmented, whereas ₁-adrenoceptor-mediated responses are not. All these results indicate that, in contrast to the arteries, in these veins ₂-adrenoceptors are situated closer to the sympathetic nerve terminals than ₁-adrenoceptors.

A similar differential location was also encountered among -adrenoceptors. ₁-Adrenoceptors that are very responsive to noradrenaline are "innervated" and mediate responses to sympathetic nerve activity, whereas ₂-adrenoceptors that are insensitive to noradrenaline are functionally "noninnervated" and function as hormone receptors for adrenaline from the adrenal medulla (Russel and Moran, 1980; Bryan et al., 1981). In electrically driven rabbit papillary muscles, it was shown that both ₁- and -adrenoceptors are located near or within the synaptic clefts of the sympathetic nerve endings (Dybvik et al., 1999). However, in the severely failing human heart, whereas ₁-adrenoceptors are apparently located close to the nerve endings, the down-regulated -adrenoceptors are situated outside the range of the neuronal influence, a fact that may have functional implications (Skomedal et al., 1998). As far as the veins are concerned, it was shown that, in normal strips of canine saphenous vein precontracted by prostaglandin F₂ in the presence of phentolamine, there was no relaxant response to either electrical stimulation or tyramine. However, in strips preloaded with adrenaline and precontracted by prostaglandin F₂ in the presence of phentolamine, electrical stimulation or tyramine caused frequency- or dose-dependent relaxations up to a maximum of 53.6% and 49%, respectively, of the steady-state precontraction (Guimarães and Paiva, 1981b). These results seem to indicate that, in this venous tissue, ₂-adrenoceptors are located relatively close to the nerve terminals, whereas ₁-adrenoceptors are not innervated (or are not abundant enough), since adrenaline coming from the nerves can efficiently cause relaxation whereas noradrenaline does not.

Apparently, the adrenoceptors under sympathetic control vary from vascular bed to vascular bed, and the vascular tone results from the simultaneous activation of receptors that are differentially influenced in the different vascular areas by the transmitters coming out from the sympathetic nerve endings. The receptors, the activation of which more importantly contribute to the basal vascular tone, are those that are "innervated": the ₁-adrenoceptors in the arterial vessels, the ₁- and ₁-adrenoceptors in the heart, and ₂- and ₂-adrenoceptors in the veins.

Noradrenaline contracts the vascular smooth muscle of most major arteries by activating postjunctional ₁-adrenoceptors (see Section III.A.1.). However, in several blood vessels, ₂-adrenoceptors also contribute to the vasoconstriction caused by noradrenaline, particularly in cutaneous arteries and veins (Polónia et al., 1985; Flavahan and Vanhoutte, 1986a). Most interesting is that the contribution of ₁- and ₂-adrenoceptors to the vasoconstriction caused by noradrenaline changes along the length of a single vessel (Bevan et al., 1980). In the arteries of the limbs, the participation of ₂-adrenoceptors for the -adrenoceptor-mediated vasoconstriction caused by noradrenaline increases from the proximal to the distal parts of these vessels. A comparison between human proximal (dorsalis pedis and arcuate arteries of the foot and superficial palmar arch of the hand) and distal arteries (digital arteries of the foot and hand) showed an increased prominence of ₂-adrenoceptors on distal, compared with proximal, arteries (Flavahan et al., 1987a). The reduction in ₂-adrenoceptor responsiveness from distal to proximal arteries continues in more proximal blood vessels such that ₂-adrenoceptor-mediated responses are not present in larger arteries of the limbs (Thom et al., 1985). In the mouse tail artery, the contractile response resulting from ₂-adrenoceptor activation is also greater at the distal than at the proximal level, whereas the opposite pattern was observed for ₁-adrenoceptors (Chotani et al., 2000).

This pattern of increasing ₂-adrenoceptor responsiveness from proximal to distal arteries is apparently not observed in the cerebral circulation. Bevan et al. (1987) showed that, in the cerebral arteries, the -adrenoceptor responsiveness becomes progressively less important with successive branching of arteries. Small branches seem to have no functional -adrenoceptors (Bevan et al., 1987). This may explain the observation that, in mice lacking the _2A/D-adrenoceptors, the pressor response to intra-arterial injection of ₂-adrenoceptor agonists (UK-14304 or dexmedetomidine) was blunted when the injection was given into the femoral artery, but not when the injection was given into the carotid artery (MacMillan et al., 1996).

In contrast to what was observed in arteries, it was shown that on isolated strips of canine saphenous and cephalic veins the maximum contractile effect of ₂-adrenoceptor activation was markedly smaller at the distal than at the proximal level; however, there was no change in the potencies of the selective ₂-adrenoceptor agonists UK-14304 or BHT 920 along the length of the vessels (Guimarães and Nunes, 1990), indicating that the density of ₂-adrenoceptors is higher at the proximal than at the distal level. This was confirmed in perfusion experiments with distal segments of canine saphenous vein where it was observed that they responded very poorly or did not respond at all to ₂-adrenoceptor agonists applied to either the intima or the adventitia (Nunes et al., 1991). Since ₂-adrenoceptors are very sensitive to changes in temperature and are abundant in cutaneous blood vessels (of the limbs in humans and dogs and of the tail in the rat) (see Section VIII.), they appear to be important for thermoregulation, by constricting on cooling and dilating when exposed to a warmer environment. It is not surprising that the distribution of ₂-adrenoceptors in cerebral and cutaneous arteries is different, since the functional role of arteries in these two beds is not comparable.

Large coronary vessels possess both - and -adrenoceptors, whereas small vessels of the coronary circulation possess only -adrenoceptors (Bohr, 1967).

-Adrenoceptors are also not uniformly distributed along the length of a single vessel. The thoracic aorta of the rabbit shows considerable -adrenoceptor-mediated activity, whereas in the abdominal aorta -adrenoceptor-mediated activity is practically nonexistent (Bevan et al., 1980). In the dog saphenous vein, the maximal relaxation to isoprenaline was much larger in the distal than in the proximal vein, whereas the effectiveness of forskolin did not vary, irrespective of the tone and the segment of vein used (Guimarães et al., 1993). Furthermore, the effectiveness of dobutamine increased from the proximal to the distal part, whereas that of the selective ₂-adrenoceptor agonist terbutaline decreased. This indicates that the effectiveness of -adrenoceptor activation and the contribution of ₁-adrenoceptors to the relaxation increase from the proximal to the distal part of the canine saphenous vein (Guimarães et al., 1993).

It is not easy to propose an explanation for this differential distribution of -adrenoceptors, since the functional consequences of their activation by any of the endogenous ligands (adrenaline and noradrenaline) is always masked by the predominant effect resulting from simultaneous activation of -adrenoceptors.

There is also a regional variation in the distribution of vascular adrenoceptors. For many years, it has been accepted, at least in humans, that splanchnic and skeletal muscle vascular beds dilate to adrenaline because -adrenoceptors predominate in their vessels, whereas adrenaline consistently reduces renal and skin blood flow, because in renal and skin vessels -adrenoceptors are predominant (for reviews, see Innes and Nickerson, 1970; Hoffman and Lefkowitz, 1995).

The cerebral and coronary arteries are of particular importance in the whole of the vascular system, because of the vital functions of the organs they supply. The cerebral circulation of many species has an abundant and dense sympathetic innervation. However, the response of the cerebral vasculature to sympathetic nerve activity is comparatively small (Bevan et al., 1980; Toda, 1983). In humans, the influence of sympathetic innervation on the tone of cerebral vasculature is weak and reflects not only a low density of innervation, but also a reduced number of -adrenoceptors (Bevan et al., 1998a). Furthermore, the sympathetic neurogenic control of cerebral arteries decreases with decreasing diameter of the vessel, such that the human pial arteries pratically do not contract in response to nerve stimulation (VanRipper and Bevan, 1991; Bevan et al., 1998a). Whereas the maximum vasoconstriction to noradrenaline in the middle meningeal artery reaches 34% of the maximum to KCl, in the pial artery it reaches only about 10% of the maximum. The cerebral arteries of the rat and pig do not contain functional -adrenoceptors (Bevan et al., 1987). There is also little evidence for a significant -adrenoceptor population in cerebral arteries (Bevan et al., 1998a). However, this relative lack of postjunctional adrenoceptors does not necessarily mean a lack of influence of the sympathetic nerves on the cerebral circulation. Some influence may be exerted through a cross-talk between sympathetic nerves and other neuronal systems. The nicotine-induced relaxation in the porcine basilar artery appears to result from the activation of nicotinic receptors on the presynaptic adrenergic nerve terminals; this activation causes release of noradrenaline that activates ₁-adrenoceptors located on NOergic nerves and promotes the release of NO (Toda et al., 1995; Zhang et al., 1998; Lee et al., 2000). Another indirect effect mediated by adenoceptors is that observed in segments of rabbit middle cerebral arteries, where the activation of endothelial ₂-adrenoceptor causes a reduction in endothelin-1 production and promotes vascular relaxation (Thorin et al., 1997).

In the coronary circulation, the relative amount of - and -adrenoceptors and the relative functional role they play also does not fit into the general pattern of the vascular beds. In the pig, the small coronary vessels exhibit little or no -adrenoceptor-mediated activity, and the large coronary artery contains ₁-adrenoceptors, mainly of the _1A-subtype, but the functional importance of their vasoconstrictive effect is unclear (Yan et al., 1998). Also in the coronary artery of the dog, the functional role of -adrenoceptors varies between undetectable and of little expression (Begonha et al., 1995). In vessels with spontaneous tone, isoprenaline causes concentration-dependent relaxations, whereas noradrenaline and adrenaline cause either contraction (of small magnitude) or relaxation. However, after the tone had been elevated by phenylephrine, both adrenaline and noradrenaline cause concentration-dependent relaxations with a maximum effect that sometimes did not fully antagonize the previous tone (Ross, 1976; Guimarães et al., 1993; Begonha et al., 1995). This is not true for isoprenaline, which, at any level of tone, causes a relaxation that totally antagonizes the previous contraction (Table 3). In contrast to the mesenteric, splenic, and pulmonary arteries, where ₂-adrenoceptors predominate, in the coronary arteries ₁-adrenoceptors are largely predominant if not exclusive (Begonha et al., 1995). In fact, whereas in the systemic arteries adrenaline was much more potent than noradrenaline as an agonist and the selective ₂-adrenoceptor antagonists ICI-118551 was much more potent than atenolol at antagonizing the responses to isoprenaline; in the coronary arteries, noradrenaline was more potent than adrenaline; and ICI-118551 and atenolol were equipotent as antagonists of isoprenaline (Begonha et al., 1995). Also in the dog, it was shown that the magnitude of -adrenoceptor-mediated responses of epicardial coronary arteries is inversely related to the size of the vessel (Krauss et al., 1992). The difference was independent of -adrenoceptors, endothelium, and second messenger processing, suggesting a mechanism based on -adrenoceptor density (Krauss et al., 1992). As far as the splanchnic vascular bed is concerned, experiments carried out on isolated mesenteric and splenic arteries showed, at least in the dog, that -adrenoceptor-mediated effects always predominate over -adrenoceptor-mediated responses when adrenaline is used as agonist (Guimarães and Paiva, 1981a; Begonha et al., 1995). It is possible that -adrenoceptors are associated only with arterioles and precapillary sphincters, which regulate the peripheral resistance observed in vivo and that are not available for studies in vitro. However, in experiments in which the hindlimb of the dog was perfused, adrenaline, despite reaching arterioles and precapillary sphincters, caused concentration-dependent increases of the perfusion pressure, showing that -adrenoceptor-mediated vasopressor effect predominates also in this vascular bed (Teixeira, 1977). An interesting peculiarity was shown in the acral regions of the cutaneous circulation, where the vascular tone is primarily controlled by humoral mechanisms mediated at postjunctional ₂-adrenoceptors; ₁-adrenoceptors that mediate neuronally evoked constriction in the cutaneous vasculature contribute little to the sympathetic regulation of this bed (Willette et al., 1991).

	VII. Influence of Maturation and Aging

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Maturation and aging are associated with many alterations in vascular adrenergic mechanisms. From birth to adulthood (maturation) and from adulthood to old age (aging or senescence), important changes occur in animal models as in humans at the receptor level, neurotransmitter process, and catecholamine inactivation.

In general terms, one can accept that maturation is associated with an increase, whereas aging is associated with a reduction in the adrenergic influence on the physiological processes.

It is well documented that the responsiveness of vascular smooth muscle to ₁-adrenoceptor activation is present at birth (Guimarães et al., 1994) and that it changes with age, although in the majority of functional studies no important alterations in responses to noradrenaline had been demonstrated either during maturation or aging (for a review, see Docherty, 1990). In the dog mesenteric artery and rat aorta, small reductions in the responsiveness to sympathomimetic amines were reported during maturation (McAdams and Waterfall, 1986; Toda and Shimizu, 1987), whereas a decrease in -adrenoceptor-mediated functions with aging was observed in the rat tail artery (Fouda and Atkinson, 1986) and in the rat aorta (Hyland et al., 1987; Wanstall and O'Donnell, 1989). According to Satoh et al. (1995), the potency of noradrenaline in the rat aorta increased with age from 3 to 10 weeks, but decreased from 10 to 40 weeks. In the pig coronary arteries, the endothelium-dependent relaxation to noradrenaline via the ₂-adrenoceptors decreases with aging (Murohara et al., 1991).

It has been suggested that the age-related changes in ₁-adrenoceptor-mediated vasoconstrictor responses in isolated blood vessels might result from changes in the expression of the ₁-adrenoceptor subtypes; accordingly, functional, radioligand binding, and molecular biology studies using rat aortic tissue have shown that with age the expression of the _1A subtype is increased, that of the _1B subtype is decreased, and that of the _1D-subtype does not change (Gurdal et al., 1995a,b). However, in the pithed rat, it was shown that the selective _1D-adrenoceptor antagonist BMY-7378 displaced the dose-response curve to phenylephrine in young prehypertensive SHRs, but had no effect in young WKY rats; whereas in adult WKY rats, BMY-7378 caused a greater shift in the concentration-response curve to phenylephrine than in younger animals (Villalobos-Molina et al., 1999). The presence of _1D-adrenoceptors in the resistance vasculature of the prehypertensive and hypertensive rats may indicate that _1D-adrenoceptors are involved in vascular hyperreactivity (Villalobos-Molina et al., 1999). This apparent contradiction may well be due to the fact that the aorta and the resistance vessels are functionally totally different. The results obtained by Xu et al. (1997) confirm that aging changes heterogeneously the expression of ₁-adrenoceptor subtypes. These authors determined the changes in mRNA levels of ₁-adrenoceptor subtypes during maturation and aging in aortae and in renal, pulmonary, and mesenteric arteries isolated from 3-, 12-, and 24-month-old rats. They observed that, in the aorta, _1A-, _1B-, and _1D-adrenoceptors declined with aging, whereas in the renal artery there was a decrease in mRNA for the _1B-adrenoceptor in aged rats. However, in mesenteric and pulmonary arteries, there were no changes in mRNA levels for any of the subtypes. The results obtained on the aggregatory responses of human platelets in radioligand binding studies also show no important differences with maturation and aging in the affinity of ligands for the binding site (Buckley et al., 1986: Davis and Silski, 1987). Vascular contractile responsiveness seems to increase with aging, and this supersensitivity may be related to the pronounced increase in the maximal pressor effect of ₁-adrenoceptor stimulation observed in the adult pithed rat (Ibarra et al., 1997). Because _1D-adrenoceptors represent the predominant subtype that mediates contraction in the aorta, carotid, and mesenteric arteries of SHRs (Villalobos-Molina and Ibarra, 1996), it may be that _1D-adrenoceptors play some role in the pathogenesis/maintenance of hypertension (Ibarra et al., 1998).

The ₂-adrenoceptor-mediated negative modulation of noradrenaline release is fully developed at birth (Guimarães et al., 1991, 1994). However, while at the postjunctional level phenylephrine is equipotent in adults and neonates, indicating that postjunctional ₁-adrenoceptors do not change during maturation, UK-14304 is about 4 times more potent at inhibiting noradrenaline release evoked by electrical stimulation in adults than in neonates (Guimarães et al., 1991). A similar difference in potency of UK-14304 at inhibiting noradrenaline release in adults and neonates was also observed in mouse atria (A.U. Trendelenburg and K. Starke, personal communication). One possible explanation for this difference is that the fractional release of noradrenaline is much higher in neonates than in adults (Guimarães et al., 1991; Moura et al., 1993). Hence, the concentration of noradrenaline in the biophase during electrical stimulation is higher in neonates than in adults; consequently, the inhibitory effect of any given concentration of UK-14304 is smaller, and its IC₅₀ is higher in neonates than in adults (Starke, 1972; Fuder et al., 1983). Based on the temporal and regional pattern of ₂-adrenoceptor mRNA expression in rat brain, it has been suggested that the perinatal increase in receptor density may serve specific roles in development, including neuronal migration, maturation of neurons, and mediation of sensory functions (Winzer-Serhan and Leslie, 1997; Winzer-Serhan et al., 1997a,b). According to Happe et al. (1999), ₂-adrenoceptors are functionally coupled to G-protein throughout postnatal development and, therefore, are able to mediate signal transduction upon stimulation by noradrenaline and adrenaline. In the neonatal rat lung, there is a pure and dense population of _2B-adrenoceptors, which is assumed to be the only one existing in this tissue. However, the number of these receptors falls to undetectable levels in adults (Latifpour and Bylund, 1983). In mice, functionally important prejunctional ₂-adrenoceptors exist in atria and vas deferens already at the age of 1 day, which are mainly _2A/D. With maturation, the _2A/D-adrenoceptors increase their functional influence. However, the development of prejunctional _2C-adrenoceptors is much more impressive. They are almost absent at birth, become influencial after birth, and reach maximum activity in adult life. In atria from adult _2A/D-adrenoceptor knockout mice, there is as much autoinhibition as in adult wild-type atria (A. U. Trendelenburg and K. Starke, personal communication).

In the canine saphenous vein, pre- and postjunctional -adrenoceptor-mediated effects are lacking at birth. However, the responses to forskolin, a direct-acting stimulant that bypasses the need for -adrenoceptors on their linkage to stimulatory G-protein subunits, are already present at birth; this shows that the lack of responses to isoprenaline is linked to either a lack or some kind of immaturity of the receptors or G-protein (Guimarães et al., 1994). Furthermore, it was shown that ₂-adrenoceptor-mediated effects and the increase in the adrenaline content of the adrenal gland have a parallel time course (Paiva et al., 1994). Thus, both the prejunctional and the postjunctional ₂-adrenoceptor-mediated effects increase with increasing age (until adulthood), as does the adrenaline content of the adrenal gland, such that at 2 weeks the ₂-adrenoceptor-mediated maximum effect is about 50% of that of the adult; and at 1 month, it is fully developed (Paiva et al., 1994). The relationship between the content of adrenaline of the adrenal medulla and the development of ₂-adrenoceptor-mediated responses was analyzed also in the rat, a species in which ₂-adrenoceptor-mediated responses develop earlier than in the dog, such that at birth these responses are already fully expressed. Interestingly, whereas the adrenaline content of the canine adrenal medulla at birth is about 3% that of the adult, in the rat it is about 50%. This suggests a link between adrenaline and the maturation of ₂-adrenoceptor-mediated effects, indicating that either adrenaline triggers the expression of ₂-adrenoceptor-mediated effects or that the expression of adrenaline formation and ₂-adrenoceptor-mediated effects are simultaneously evoked by the same event (Moura et al., 1997). Similarly, in the mouse, a species in which adrenaline represents about 60% to 70% of total catecholamines in the adrenal medulla of 1-day-old animals, prejunctional ₂-adrenoceptors fully operate at that age (A.U. Trendelenburg and K. Starke, personal communication). This hypothesis is in good agreement with the report that in early neonatal life isoprenaline, instead of producing desensitization of responses, enhances expression or efficiency of -adrenoceptor signaling (Giannuzzi et al., 1995). Recently, it was demonstrated that agonist treatment in the neonate causes an enhancement of coupling rather than an uncoupling of receptors from G-proteins (Zeiders et al., 1999), and the reversal from enhancement of coupling to uncoupling occurs (in cardiac cells) between postnatal days 11 and 14 (Zeiders et al., 2000).

-Adrenoceptor-mediated relaxation was compared in the pulmonary vein of the fetal (145 ± 2 days of gestation) and newborn lamb. Isoprenaline caused greater relaxation in newborn than in fetal lambs. Also, in humans, the sympathetic nerves play a more important role in the regulation of cerebrovascular tone in the infant than in the adult (Bevan et al., 1998b). Biochemical studies showed that isoprenaline and forskolin evoked a greater increase in cAMP content and in adenyl cyclase activity of pulmonary veins in the newborn than in the fetal lamb. These results show that -adrenoceptor-mediated relaxation of the pulmonary veins increases with maturation (Gao et al., 1998). However, according to Conlon et al. (1995), there is no change in myocardial ventricle -adrenoceptor G-protein coupling capacity or adenylate activation with aging beyond maturity. These authors showed that aging between 6 and 26 months in male Wistar rats is not accompanied by changes in myocardial -adrenoceptor signal transduction and capacity for formation of the high-affinity -adrenoceptor G-protein coupled complex with the agonist. It was also found that an age-related impairment of myocardial -adrenoceptor up-regulation occurs with aging (Conlon et al., 1995).

This -adrenoceptor-mediated relaxing capacity, which increases during the first weeks of life, then declines as the age increases. The loss of vasodilator response to isoprenaline in the rat aorta has been reported at different ages ranging from 3 to 22 months (for a review, see Docherty, 1990). There is not only a decrease in the maximum relaxation to isoprenaline with aging, which has been reported for the rabbit aorta, rat pulmonary artery, rat mesenteric artery, human saphenous vein, canine mesenteric artery, but also an increase in the EC₅₀ of isoprenaline: in the aorta of 5- and 20-week-old rats preconstricted with phenylephrine, the pD₂ values for isoprenaline were 7.97 and 6.57, respectively (Borkowski et al., 1992), indicating a marked reduction in the potency of this -adrenoceptor agonist. According to Dohi et al. (1995), with increasing age, maximum -adrenoceptor-mediated relaxation decreases in most arteries, but not in veins. Also SHRs exhibit an age-related loss in vasodilator -adrenoceptor responsiveness. However, the maximum relaxation to sodium nitrite or to sodium nitroprusside is not reduced (O'Donnell and Wanstall, 1986; Küng and Lüscher, 1995).

Because most studies show no change with age in the number of ₁- or ₂-adrenoceptor-binding sites of the human lymphocytes and rat heart and because cAMP production in response to forskolin and dibutyril cyclic adenosine monophosphate is also reduced by aging in the rat myocardium and human lymphocytes, it seems likely that the change is not at the receptor level but in the coupling to the adenylate cyclase via G-proteins. In healthy volunteers of different ages, isoprenaline-induced increases in heart rate were significantly greater in young than in old ones (Brodde et al., 1998). However, -adrenoceptor numbers and subtype distribution were unchanged as determined in patients undergoing open heart surgery. The decrease in -adrenoceptor-mediated efficiency is due to a reduced activity of the catalytic unit of the adenylyl cyclase (Brodde and Pönicke, 1998).

Prolonged or repeated exposure to -agonists in adults results in a compensatory desensitization that reduces responsiveness (for a review, see Summers et al., 1997). In older animals, the predominant effect is heterologous desensitization mediated at the level of the G-protein. During development, however, responses in most systems increase with age and with the maturation of neuronal inputs (Giannuzzi et al., 1995). Instead of producing desensitization of responses, agonist exposure promotes receptor signaling by enhancing expression and/or catalytic efficiency of adenylyl cyclase. These developmental differences are likely to be important in the maintenance of tissue responsiveness during the period in which innervation develops (Guimarães et al., 1994; Giannuzzi et al., 1995; Moura et al., 1997).

Regarding the age-related involvement of the endothelium in -adrenoceptor-mediated responses, it has been shown that aging reduced endothelium-dependent relaxations to acetylcholine and isoprenaline in aortas from both normotensive and SHR rats (Arribas et al., 1994; Küng and Lüscher, 1995; Satake et al., 1995; van der Zypp et al., 2000). However, the responses to the NO-donor nitroprusside sodium, which directly activates the soluble guanylyl cyclase and formation of cGMP, were very similar in adult and old rats of either strain. This indicates that the impairment of the response to acetylcholine and isoprenaline is due to functional changes of the endothelium, rather than the vascular smooth muscle (Küng and Lüscher, 1995; van der Zypp et al., 2000). Furthermore, it was observed that, in endothelium-intact aortas, the nitric oxide synthase inhibitor L-NMMA attenuated the isoprenaline-induced relaxation to a similar extent in both age groups, suggesting that although NO was involved in the response to isoprenaline, it cannot have been responsible for the age-related difference (van der Zypp et al., 2000). It was also observed that the age-dependent reduction in isoprenaline-mediated relaxation in aorta was greater in K⁺ than in phenylephrine-constricted aortas (Borkowski et al., 1992; Chapman et al., 1999), supporting the view that the signaling pathway involved in isoprenaline-induced relaxation switches toward an increased role of K⁺ channels in older rats. Thus, the signaling pathways involved in -adrenoceptor-mediated responses are multifactorial. They include a NO-dependent pathway, that does not depend on age; an endothelium-independent pathway involving cAMP, which appears to decline with age; and a third factor apparently endothelium-dependent that involves tetraethylammonium-sensitive K⁺ channels and increases with age (Satake et al., 1996, 1997; van der Zypp et al., 2000).

	VIII. Influence of Temperature on Vascular Adrenoceptor-Mediated Responses

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It is common knowledge that cold makes the skin pale and heat makes the skin red. In intact organisms, exposure to cold causes cutaneous veins to constrict, whereas deeper veins dilate thus transferring venous blood from the superficial to the deep circulation to reduce heat loss (Vanhoutte, 1980). In isolated veins of the dog contracted with exogenous noradrenaline or sympathetic nerve stimulation, cooling enhances (saphenous) or reduces (femoral) the contractile responses (Vanhoutte and Lorenz, 1970). Furthermore, it was reported that cooling enhances the contractile response to the selective ₂-adrenoceptor agonist UK-14304 (Flavahan and Vanhoutte, 1986b; Nunes and Guimarães, 1993), does not change that to the selective ₁-adrenoceptor agonist phenylephrine (Flavahan and Vanhoutte, 1986b; Nunes and Guimarães, 1993), and markedly reduces the response to chloroethylclonidine (Nunes and Guimarães, 1993). Similarly, cooling enhanced the ₂-adrenoceptor-mediated contractile effect evoked by electrical stimulation on the human saphenous vein (Harker et al., 1994). Flavahan and Vanhoutte (1986b) explained the different sensitivity of cutaneous and deep blood vessels to cooling on the basis of a different ₁-adrenoceptor reserve: in the saphenous vein, an enhanced ₂-adrenoceptor-mediated effect is added to a nonreduced ₁-adrenoceptor-mediated response (because in this vein there is a large ₁-adrenoceptor reserve that buffers the ₁-adrenoceptor-mediated response from the inhibitory influence of cooling); in the femoral vein, the ₂-adrenoceptor-mediated effect is so inefficient that its enhancement does not compensate for the markedly reduced ₁-adrenoceptor-mediated responses (which is depressed because there is no ₁-adrenoceptor reserve). The same authors had previously shown in the saphenous vein that under normal conditions, cooling to 24°C did not affect the responses to phenylephrine, whereas it did reduce markedly this response after partial irreversible blockade of ₁-adrenoceptors with phenoxybenzamine (Flavahan and Vanhoutte, 1986b). However, the enhancement of the ₂-adrenoceptor-mediated responses remained to be explained.

In the deer digital arteries, a different reactivity was found in winter and summer: in the cold winter, they were either insensitive or had a reduced sensitivity to the vasodilator action of histamine, compared with arteries collected in summer (Callingham et al., 1998; Milton et al., 1999).

Very recently, it was shown that, in the mouse tail artery, at 37°C, vasoconstriction to the ₂-adrenoceptor agonist UK-14304 was antagonized by the selective _2A/D-adrenoceptor antagonist BRL-44408, but was not antagonized by the _2B- and _2C-adrenoceptor antagonist ACR-239 or the preferential _2C-adrenoceptor antagonist MK-912. However, at 28°C, the enhanced vasoconstrictor response to UK-14304 was inhibited by low concentrations of the preferential _2C-adrenoceptor antagonist MK-912, whereas ACR-239 was ineffective and the selective _2A/D-adrenoceptor antagonist BRL-44408 showed an inhibitory effect that was not different from that observed at 37°C. These results indicate that, at 28°C, _2C-adrenoceptors contribute to ₂-adrenoceptor-mediated vasoconstriction and probably are responsible for the supersensitivity to ₂-adrenoceptor agonists caused by cold (Chotani et al., 2000). Interestingly, this is not a phenomenon exclusively occurring with vascular smooth muscle of superficial vessels, since hypothermia enhanced ₂-adrenoceptor-mediated responses in rat vas deferens in such a way that the lack of any response to UK-14304 at 37°C was converted to evident contractions at 20°C (Gonçalves et al., 1989).

	IX. Vascular Adrenoceptors in Some Diseases

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Vascular adrenoceptors may be affected in many diseases, sometimes as a consequence of alterations suffered by the vessels and sometimes by participating themselves in the genesis of diseases or by being their primary cause.

For many diseases, there are no animal models. Even in well studied animal models, such as the SHRs, relevance to human "essential hypertension" is unknown. As a consequence, one often has to rely on observations with patients. Additionally, in vivo experiments in animals (e.g., measurements of blood pressure) often involve multifactorial systems, the analysis of which is far more complex than in the in vitro experiments. For instance, the determination of maximum responses of the blood pressure to pressor agents is often impossible; hence, it is difficult or impossible to provide a full and satisfactory description of an "enhanced pressor response" observed in this or that disease: is this phenomenon due to a parallel shift of the dose-response curve to the left, to an increase of the maximum response, or to both? Is this phenomenon generated by a change in the structure of the blood vessels by a change in the mechanisms that inactivate the test agonist, by a change in G-proteins or second messenger mechanisms, or in change of the adrenoceptors? Given these unavoidable limitations of attempts to delineate the role played by adrenoceptors in various diseases, it is not surprising that hard facts are rare. However, such studies have provided valuable hints.

In 1929, Lewis postulated that Raynaud's disease resulted from a "local fault" of the blood vessel wall. This fault could be an anomalous regulation of ₂-adrenoceptors. It appears now clear that ₂-adrenoceptors play a role in the development of Raynaud's disease (Freedman et al., 1995; Chotani et al., 2000). Cold augments constriction to ₂-adrenoceptor activation without affecting the responses to ₁-adrenoceptor stimulation or to any other vasoconstrictor agent (Flavahan and Vanhoutte, 1986a; Chotani et al., 2000). Nonselective ₂-adrenoceptor antagonists abolish cold-induced vasospastic crises in patients with primary Raynaud's disease (Freedman et al., 1995). However, according to many authors, there is no abnormal reactivity of ₂-adrenoceptors in subjects with primary Raynaud's disease (Lindblad et al., 1989; Coffman and Cohen, 1990; Freedman et al., 1993). Therefore, the local fault in Raynaud's crisis may represent a consequence of a cold-induced functional expression of _2C-adrenoceptors that appear to be silent at normal temperature (Chotani et al., 2000). Thus, inhibition of _2C-adrenoceptors may provide a highly selective therapeutic measure for this disease.

Vasospasm and ischemic organ injury are functional changes that play an important role in the pathogenesis of scleroderma (Kahaleh, 1990). These functional changes were attributed to a failure of vascular endothelium in releasing nitric oxide (Freedman et al., 1999). However, in arterioles isolated from uninvolved skin of patients with scleroderma, the constrictor responses to the selective ₂-adrenoceptor agonist UK-14304 were increased, whereas those to KCl or the selective ₁-adrenoceptor agonist phenylephrine were similar to controls. This selective increase in the reactivity of ₂-adrenoceptors was not altered by removing the endothelium, indicating that the enhanced constrictor effect was not due to changes in endothelial dilator activity, but to an enhancement of the ₂-adrenoceptor-mediated responses of the vascular smooth muscle cells (Flavahan et al., 2000).

In several neurological degenerative and genetic disorders, there are also important changes in -adrenoceptor-mediated responses. In the majority of these situations, the pathological process involves primarily the sympathetic postganglionic neurones, leading to a progressive denervation of some organs, including the blood vessels. The most prominent cardiovascular symptom in all these conditions is orthostatic hypotension, which is a common complaint due to a sympathetic neurocirculatory failure and sometimes forces the patients to be bedridden, even if they are still able to work. In the familial amyloidotic polyneuropathy, an autosomal dominant disorder with an estimated prevalence of about 1/1000 in the population of the most affected areas in the northwest of Portugal (Andrade, 1952; Carvalho et al., 1997), there is a progressive degeneration of the sympathetic nerves leading to complete denervation. Concomitantly, there is a marked supersensitivity to the vasoconstrictor action of noradrenaline (Falcão-de-Freitas, 1996; Carvalho et al., 1997), probably due to an up-regulation of -adrenoceptors. It is not yet known whether all -adrenoceptor subtypes are equally involved or some particular subtype(s) is predominantly implicated. A similar enhancement of -adrenoceptor-mediated responses occurs in patients with congenital dopamine-hydroxylase deficiency (Man in't Veld et al., 1987). In this disorder, there is no conversion of dopamine to noradrenaline causing a lack of the transmitter at postganglionic sympathetic neurones (Man in't Veld et al., 1987; Rea et al., 1990). Recently, it was shown that many patients with Parkinson's disease have evidence of peripheral sympathetic denervation causing a deficient release of noradrenaline in the heart and blood vessels with a consequent -adrenoceptor up-regulation (Magalhaes et al., 1995; Netten et al., 1995; Goldstein et al., 2000). In diabetic polyneuropathy, there is also an autonomic dysfunction leading to a progressive sympathetic denervation and to a more or less marked increase in -adrenoceptor-mediated responses (for review, see Watkins, 1998). There are other degenerative neurological diseases, like the multiple system atrophy (Shy Drager syndrome), in which the main pathological changes leading to the neurocirculatory failure with severe orthostatic hypotension occur in the central nervous system without marked alterations of the peripheral sympathetic nerves (Zoukos et al., 1999; Goldstein et al., 2000). Very recently, it was shown that overexpression of the _1B-adrenoceptor causes apoptotic neurodegeneration with a corresponding multiple system atrophy (Zuscik et al., 2000). The resulting symptoms (impaired hindlimb function and seizures) could be rescued with the ₁-adrenoceptor antagonist terazosin, indicating that ₁-adrenoceptors participated directly in the pathology. These findings suggest a link between _1B-adrenoceptor function and the etiology of Shy-Drager syndrome (Zuscik et al., 2000).

Sometimes, -adrenoceptors are also involved in these neurological degenerative diseases. Both in multiple sclerosis and multiple system atrophy, there is sympathetic neurocirculatory failure with supersensitivity to -adrenoceptor agonists. However, whereas in multiple sclerosis there is also an up-regulation of the -adrenoceptors expressed on peripheral blood mononuclear cells, in multiple system atrophy there is not (Zoukos et al., 1999).

According to many authors, -adrenoceptors may be involved in the pathogenesis/maintenance of some kinds of hypertension. Not only sensitivity to salt is a common trait in patients with essential hypertension, but there is also experimental evidence suggesting that salt loading causes hypertension via a mechanism involving ₂-adrenoceptors. A recent comparison of the effect of subtotal nephrectomy and salt loading in _2B-adrenoceptor knockout mice, in _2C-adrenoceptor knockout mice, and in wild-type mice showed that only the _2B-adrenoceptor knockout mice have no significant increase in blood pressure (Makaritsis et al., 1999). Both the wild-type and _2C-adrenoceptor knockout mice had significant blood pressure increases, indicating that _2B-adrenoceptors are relevant for the development of this kind of hypertension. On the other hand, some data draw attention to the possible role played by changes in ₁-adrenoceptors in the development/maintenance of hypertensive states. In the resistance vasculature of young prehypertensive and hypertensive rats, a high density of _1D-adrenoceptors was found (Stassen et al., 1997; Ibarra et al., 1998; Xu et al., 1998). Furthermore, in endothelium-denuded tail artery and aorta, the maximum contractile response to phenylephrine and chloroethylclonidine was higher in SHR than in WKY rats (Villalobos-Molina et al., 1999; Ibarra et al., 2000), suggesting that _1D-adrenoceptors may be involved in vascular supersensitivity leading (or in some way being linked) to the hypertensive state. It is also possible that changes at postadrenoceptor level, such as altered levels of G-proteins may be related to the higher reactivity to agonists in hypertension (Li et al., 1994; Kanagy and Webb, 1996). These results indicate that future studies with _1D-adrenoceptor knockout animals may be helpful to explain the role (if any) of these alterations in genesis/maintenance of hypertension.

Trp64Arg mutation of ₃-adrenoceptor has been suggested to confer susceptibility to essential hypertension (Morris et al., 1994); this thesis was contested by Fujisawa et al. (1997) and confirmed by Tonolo et al. (1999). These authors concluded that the Trp64Arg polymorphism of the ₃-adrenoceptor gene is associated more often with high blood pressure than with normal blood pressure.

A naturally occurring variation found in about 8% of Europeans and North Americans actually restores in humans the arginine residue present in animals (Strosberg, 1997). This variation was found to be associated with 1) an increased capacity of obese French patients to gain weight (Clément et al., 1995); 2) an early onset of noninsulin-dependent diabetes mellitus in obese Pima Indians (Walston et al., 1995); and 3) an early onset of noninsulin-dependent diabetes mellitus and clinical features of the insulin resistance syndrome in Finns (Widén et al., 1995). Although these alterations are related to ₃-adrenoceptors in adipocytes, they may be particularly important since it is believed that in terms of the risks of cardiovascular disorders, visceral obesity is the most dangerous form of regional fat accumulation, the form of obesity that is more directly linked to ₃-adrenoceptor activity (Arner, 1995).

	X. Conclusions

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New possibilities are now offered by molecular biology (knockout animals, genetically altered receptors, measurements of mRNA, etc.) that will help in clarifying receptor function. However, data obtained in experiments carried out in knockout animals must be carefully interpreted, keeping in mind the multiple ways to compensate for the lack of this (or these) adrenoceptor subtype(s).

Regarding the subclassification of adrenoceptors, two controversial points are now on the way to being solved: the existence of a fourth ₁-adrenoceptor subtype (the _1L-adrenoceptor) and a fourth -adrenoceptor (the ₄-adenoceptor). It is now accepted that these hypothetical subtypes correspond to low-affinity states of the _1A- and the ₁-adrenoceptors, respectively.

The pharmacology of human ₁-adrenoceptors often differs from that of the corresponding ₁-adrenoceptor subtypes of experimental animals. Then, the identification of ₁-adrenoceptor subtypes present in human vasculature may be useful for the discovery of new selective compounds effective in the treatment of prostatic hypertrophy, pulmonary hypertension, and coronary insufficiency.

A fascinating point is that, in the vast majority of the organs, the adrenoceptors expressed there do not correspond to the functional roles they play. The potential role of some adrenoceptor subtypes apparently unimportant under normal conditions, should be kept in mind and carefully taken into consideration. It has been described that some sodium channels (II and III -isoforms), which are functionally important during the earlier stages of life, loose their important roles in adult life and reappear functionally active under some pathological conditions (Aronica et al., 2001). This fact should be linked to the interesting fact that expression and function of a given adrenoceptor subtype changes their role during a lifetime. For example the _2B-adrenoceptor, which is densely represented during intrauterine development, disapears after birth, whereas the _1D-adrenoceptors that have no important role under physiological conditions may become important in some hypertensive states. This dynamic balance can also be exemplified by the fact that _2C-adrenoceptors, which at 37°C are nonfunctional, become functionally predominant at lower temperatures.

Prejunctional _2A/D-adrenoceptors are now well established to be primarily responsible for the regulation of the release of noradrenaline under physiological conditions. However, also, _2C-adrenoceptors play a minor role in this regulatory mechanism.

The existence of - and -adrenoceptors in the endothelium and the importance of the endothelial system in the physiology and pathophysiology of the vascular system has to be considered; however, it is intriguing that the activation of - and -adrenoceptors lead to the same effect: an increase in NO formation/release.

From a therapeutic standpoint (and as far as -adrenoceptors are concerned), there are many instances where -adrenoceptor-subtype selective stimulation (asthma, atrioventricular block, obesity) or block (hypertension, coronary insufficiency) is desired. Therefore, a still more detailed knowledge of subtype-specific functions is necessary as drugs, which are more selective, are required.