I. Introduction

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Cardiac function is controlled by the autonomic nervous system (i.e., the sympathetic and the parasympathetic nervous systems), which act via adrenoceptors and muscarinic acetylcholine receptors, respectively. At least nine adrenoceptor subtypes and five muscarinic receptor subtypes exist. In recent years, it has been attempted to associate individual cardiac functions with individual receptor subtypes to further physiological understanding and to identify potential targets for a more specific drug treatment of cardiac disease. The autonomic control of cardiac function can be dynamically regulated by physiological factors such as aging and by disease states such as congestive heart failure. Moreover, interindividual differences may exist due to receptor gene polymorphisms. This article reviews the presence and function of adrenoceptor and muscarinic receptor subtypes in the human heart, as well as their physiological and pathophysiological regulation. Insights into autonomic control of cardiac function from receptor knockout and transgenic animals also are discussed.

	II. Presence and Function of Receptor Subtypes in Human Heart

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Three subtypes of ₁-adrenoceptors have been identified pharmacologically and through molecular cloning: _1A (formerly _1c), _1B, and _1D (formerly _1a/d; Fig. 1; Hieble et al., 1995; Michel et al., 1995). They are encoded by distinct genes that are located on human chromosomes 8, 5q33, and 20p13, respectively. Further ₁-adrenoceptor heterogeneity is generated by the existence of splice variants of the _1A-adrenoceptor, which differ in their length and sequences of the C-terminal domains (Hirasawa et al., 1995; Chang et al., 1998). All four splice variants can be translated into functional, phospholipase C (PLC)²-coupled receptors and have a similar pharmacological recognition profile in competition radioligand binding studies on transfection into Chinese hamster ovary cells (Hirasawa et al., 1995). Because the C-terminal domain of the ₁-adrenoceptors contains amino acids that are believed to be important for receptor desensitization processes (Lattion et al., 1994), it can be speculated that the splice variants of the _1A-adrenoceptor may exhibit differential susceptibility to down-regulation, but this has not been tested experimentally. Some studies have proposed the existence of a fourth ₁-adrenoceptor subtype that is primarily characterized by a relatively low affinity for prazosin and some other compounds and therefore was designated _1L (Muramatsu et al., 1990). However, despite extensive efforts, this putative subtype has not been cloned. Recent evidence suggests that it may be a functional state of the _1A-adrenoceptor (Ford et al., 1997). The pharmacological characteristics of ₁-adrenoceptor subtypes are shown in Table 1.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Adrenoceptor subtypes.

View this table: [in this window] [in a new window]   TABLE 1 Pharmacological characterization of 1-adrenoceptor subtypes

In the human heart, the presence of ₁-adrenoceptors was examined at the mRNA level through RNase protection assays and reverse transcription-polymerase chain reaction (RT-PCR). mRNA for the _1B-adrenoceptor has been detected with RT-PCR (Ramarao et al., 1992; Faure et al., 1995), and small amounts were seen in RNase protection assays in one (Price et al., 1994b) but not another (Weinberg et al., 1994) study. Similarly, mRNA for the _1D-adrenoceptor was found with RT-PCR (Faure et al., 1995) and in RNase protection assays in one (Price et al., 1994b) but not another (Weinberg et al., 1994) study. However, all available studies agree that the _1A-adrenoceptor is the most abundant ₁-adrenoceptor subtype in the human heart at the mRNA level (Hirasawa et al., 1993; Price et al., 1994b; Weinberg et al., 1994; Faure et al., 1995). Moreover, all three splice variants of the _1A-adrenoceptor exist in the human heart, although with differing abundance (Hirasawa et al., 1995; Chang et al., 1998). The functional relevance of the _1A-adrenoceptor splice variants in the human heart, as in other tissues, remains to be tested. Although little is known about a possible differential distribution of ₁-adrenoceptor subtypes in various parts of the human heart, studies in rats indicate that the relative abundance of the three subtypes at the mRNA level is similar in all parts of the heart (Wolff et al., 1998). However, it should be noted that an extrapolation from the rat to the human heart may not be possible because _1B- rather than _1A-adrenoceptors have the greatest relative abundance in rat heart (Price et al., 1994a; Wolff et al., 1998) and because the overall expression of functional cardiac ₁-adrenoceptors in rats is much greater than that in humans (see below).

Although the presence of mRNA can predict the presence of corresponding protein in many cases, this has not always been the case with regard to ₁-adrenoceptor subtypes. Thus, the detection of ₁-adrenoceptor protein in the human heart by radioligand binding studies has not been easy despite the presence of large amounts of corresponding mRNA. Nevertheless, several groups of investigators have detected small numbers of human cardiac ₁-adrenoceptors in the right and left ventricles of the human heart (Böhm et al., 1988; Bristow et al., 1988; Limas et al., 1989a; Vago et al., 1989; Steinfath et al., 1992a,b; Hwang et al., 1996). Whenever ₁- and -adrenoceptor densities were compared within the same study, the latter was always by far more abundant (Böhm et al., 1988; Bristow et al., 1988; Steinfath et al., 1992b; Fig. 2). In a direct comparative study, the human right and left ventricles had the smallest ₁-adrenoceptor density among seven species, including rat, guinea pig, mouse, rabbit, pig, and calf; in contrast, rats had the highest cardiac ₁-adrenoceptor density, which exceeded that of all other species by at least 5-fold (Steinfath et al., 1992a; Fig. 3). These data indicate that the high ₁-adrenoceptor density in rat ventricles may be a particular feature of that species and necessitate great care in extrapolation of rat data to the human heart. Although the contribution of individual subtypes to the overall ₁-adrenoceptor density has been studied [e.g., in rat heart (Gross et al., 1988; Knowlton et al., 1993; Michel et al., 1994a) and rabbit heart (Endoh et al., 1992; Hattori et al., 1996)], little is known in this regard for the human heart at the protein level.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Ventricular 1- and -adrenoceptors (ARs) in the nonfailing human heart. Left, number of -adrenoceptors and 1-adrenoceptors (in fmol/mg protein). Right, positive inotropic effect of isoprenaline (via -adrenoceptor stimulation) and phenylephrine (in the presence of 1 µM propranolol via 1-adrenoceptor stimulation) in isolated electrically driven ventricular trabeculae (in  mN). Numbers at the bottom of the columns (left) and in parentheses (right) indicate number of experiments. Data for 1-adrenoceptor number are recalculated from Bristow et al. (1988), Böhm et al. (1988), and Steinfath et al. (1992a,b), and data for the positive inotropic effects of phenylephrine are recalculated from Böhm et al. (1988) and Steinfath et al. (1992b). Data for -adrenoceptor number are recalculated from Bristow (1993), Brodde et al. (1998b), and Steinfath et al. (1992b), and data for the positive inotropic effects of isoprenaline are recalculated from Brodde et al. (1998b) and Steinfath et al. (1992b).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Species dependence of cardiac 1-adrenoceptor number, which was assessed by [3H]prazosin binding in ventricular membranes. Numbers at the bottom of the columns indicate number of experiments. Data are recalculated from Steinfath et al. (1992a).

The functional role of ₁-adrenoceptors has been studied extensively in the nonhuman mammalian heart, and several review articles have been published on this topic (Endoh, 1991; Benfey, 1993; Terzic et al., 1993; Hein and Kobilka, 1997; Li et al., 1997a). Cardiac ₁-adrenoceptors can couple to numerous intracellular signal transduction responsesnot only PLC and phospholipase D but also various ion currents, including L-type Ca²⁺ channels, the transient outward current I_to, the delayed rectifier K⁺ current, and the acetylcholine-activated K⁺ channel (I_{K Ach}); moreover, the Na⁺/H⁺ exchanger and Na⁺,K⁺-ATPase can be activated (Endoh, 1991). In transgenic mice overexpressing the wild-type (WT) hamster _1B-adrenoceptor by more than 40-fold (Akhter et al., 1997a) or expressing a constitutively active mutant thereof (Milano et al., 1994b), myocardial diacylglycerol content was increased, indicating a tonic activation of the PLC pathway by cardiac ₁-adrenoceptors in vivo. The relevance of this finding, however, is unclear because the physiological expression of ₁-adrenoceptors, largely representing the _1B-adrenoceptor, is low in murine heart (Yang et al., 1998), and _1B-adrenoceptor knockout mice do not show overt signs of an altered cardiovascular function (Cavalli et al., 1997). Stimulation of cardiac ₁-adrenoceptors does not lead to elevation of cellular cAMP content (Schümann et al., 1975; Brodde et al., 1978; Wagner and Brodde, 1978; Bogoyevitch et al., 1993) but if anything may even reduce cardiac cAMP content. This inhibition may relate to coupling to pertussis toxin (PTX)-sensitive G proteins of the G_i family rather than to activation of cAMP-degrading phosphodiesterases (Barrett et al., 1993). Accordingly, WT _1B-adrenoceptor-overexpressing mice had mitigated inotropic responses to -adrenergic stimulation, which were apparently due to _1B-adrenoceptor coupling to PTX-sensitive G proteins resulting in adenylyl cyclase inhibition, and to up-regulation of the G protein-coupled receptor kinase 2 (GRK2, formerly named -adrenergic receptor kinase; Akhter et al., 1997a). More distal signaling events after the stimulation of cardiac ₁-adrenoceptors involve the activation of protein kinase C (PKC; Clerk and Sugden, 1997), of which the -, ₁,- ₂-_, ,- -, /-, and -isoforms are present in human heart (Erdbrügger et al., 1997; Bowling et al., 1999), and the extracellular signal-regulated (Thorburn and Thorburn, 1994; Bogoyevitch et al., 1996; Post et al., 1996; Zechner et al., 1997) and p38 forms of the mitogen-activated protein kinases (Zechner et al., 1997; Clerk et al., 1998). The JNK forms of the mitogen-activated protein kinases, however, are not consistently activated on stimulation of cardiac ₁-adrenoceptors (Zechner et al., 1997). ₁-Adrenoceptor signal transduction results in enhanced inotropy under most conditions, but the net effect of the various signaling responses may also be a reduced force development secondary to PKC activation (Kissling et al., 1997; Peters et al., 1998). The mechanism of the positive inotropic effect induced by ₁-adrenoceptor stimulation is still a matter of debate: ₁-adrenoceptor stimulation causes formation of 1,4,5-inositoltrisphosphate and diacylglycerol, with the former mediating release of Ca²⁺ from intracellular stores, which might be involved in increases in force of contraction. In addition, ₁-adrenoceptor stimulation increases the Ca²⁺ sensitivity of myofilaments and the transsarcolemmal Ca²⁺ influx and causes intracellular alkalinization via activation of the Na⁺/H⁺ exchanger; it has been suggested that these effects are, at least in part, due to diacylglycerol-induced activation of PKC (Endoh, 1991; Terzic et al., 1993).

Apart from these short-term effects, extended stimulation of cardiac ₁-adrenoceptors may also cause the development of a hypertrophic phenotype. This has been studied in depth in cultured neonatal rat cardiomyocytes (Meidell et al., 1986; Lee et al., 1988; Waspe et al., 1990) but may also occur in cultured cardiomyocytes obtained from adult rats (Ikeda et al., 1991; Schlüter and Piper, 1992; Pinson et al., 1993) or even in rats in vivo (Zierhut and Zimmer, 1989). Whether these findings are applicable to other species is unclear because transgenic mice overexpressing WT _1B-adrenoceptors by more than 40-fold did not develop signs of cardiac hypertrophy despite an 8-fold increase in ventricular mRNA for atrial natriuretic peptide (Akhter et al., 1997a). Only transgenic mice with cardiac overexpression of a constitutively active _1B-adrenoceptor exhibited increased myocardial atrial natriuretic peptide mRNA and considerable cardiac hypertrophy (Milano et al., 1994b). Although it can be argued that mice have only a very low cardiac expression of ₁-adrenoceptor protein, this also is true for the human heart (Steinfath et al., 1992a; Yang et al., 1998; see Fig. 3). Whether activation of ₁-adrenoceptors in the human heart promotes cardiac hypertrophy is unknown. Therefore, molecular pathways leading to ₁-adrenoceptor-stimulated cardiac hypertrophy in rats are only shortly summarized, and the reader is referred to several recent reviews on this topic for more details (Bogoyevitch and Sugden, 1996; Force et al., 1996; Page and Doubell, 1996; Olson and Molkentin, 1999; Sugden, 1999). ₁-Adrenoceptor-stimulated cardiac hypertrophy in rats occurs predominantly, if not exclusively, via _1A-adrenoceptors (Knowlton et al., 1993; Autelitano and Woodcock, 1998), which is the subtype that dominates in the human heart, at least at the mRNA level (see above). These receptors act via the PTX-insensitive G protein G_q (LaMorte et al., 1994), which in turn leads to activation of PLC (Knowlton et al., 1993) and PKC (Shubeita et al., 1992; Karns et al., 1995; Bogoyevitch et al., 1996). Accordingly, transgenic cardiac overexpression of G_q (D'Angelo et al., 1997; Adams et al., 1998; Mende et al., 1998; Dorn et al., 1999) or transfection of cardiomyocytes with a constitutively active form of G_q (Adams et al., 1998) or PKC, PKC, PKC, or PKC causes cardiac hypertrophy (for reviews, see Bogoyevitch and Sugden, 1996; Simpson, 1999). On the other hand, cardiac overexpression of a construct to inhibit G_q function (a carboxyl-terminal peptide of the -subunit of G_q) can inhibit overload-induced myocardial hypertrophy (Akhter et al., 1998). Distal to G_q activation, several signal-transducing molecules have been implicated in the development of ₁-adrenoceptor-stimulated cardiac hypertrophy, but the exact chain of events, and particularly the interaction between these mediators, remains a matter of debate (Bogoyevitch and Sugden, 1996; Force et al., 1996; Page and Doubell, 1996; Olson and Molkentin, 1999; Simpson, 1999; Sugden, 1999). Such mediators include the small GTP-binding proteins ras and rho, PKC, the extracellular signal-regulated and p38 members of the family of mitogen-activated protein kinases, and the protein phosphatase calcineurin. The latter is of particular interest for two reasons (for review, see Olson and Molkentin, 1999; Sugden, 1999): First, calcineurin may be an integrator and common mediator of several pathways leading to cardiac hypertrophy. Accordingly, calcineurin inhibition might prevent development of cardiac hypertrophy. This has in fact been shown in some animal models in vivo; however, several negative studies have also been published (for an overview, see Walsh, 1999). Second, inhibitors of calcineurin function (i.e., the immunosuppressant drugs cyclosporin A and tacrolimus, also known as FK506) are available that can be used to test hypotheses regarding the role of calcineurin in humans. However, the interpretation of the emerging human data is complicated by nephrotoxic effects of these drugs. Moreover, studies in humans are hampered by the fact that tissue material for investigation usually is available only from patients with late stages of disease processes, and some mediators relevant for cardiac hypertrophy induction may already have returned to baseline levels or even disappeared in those stages. Therefore, it remains unclear whether and how cardiac hypertrophy can develop secondary to myocardial ₁-adrenoceptor stimulation in humans.

The multitude of studies on the signal transduction of rat cardiac ₁-adrenoceptors is sharply contrasted by the very limited information that is available regarding the signal transduction of human cardiac ₁-adrenoceptors. Thus, noradrenaline stimulation (in the presence of propranolol) of human atrial appendages causes a breakdown of phosphatidylinositolbisphosphate as demonstrated by the rapid and time-dependent formation of inositol-1,3-bisphosphate, inositol-4-phosphate, inositol-1,4-bisphosphate, and inositol-1,4,5-trisphosphate (Bristow et al., 1988; Anderson et al., 1995). However, the extent of PLC activation in those studies was much less than that in rat atria studied under the same conditions, which is as to be expected based on the much smaller ₁-adrenoceptor density in the human heart (see Fig. 3).

It is generally assumed that ₁-adrenoceptors couple to their signal transduction machinery primarily via PTX-insensitive G proteins of the G_q/11 family (Bylund et al., 1994; Graham et al., 1996). Based on studies with rats, rabbits, and dogs, this also appears to be the case for many responses in the heart (Han et al., 1989; Steinberg et al., 1989; Del Balzo et al., 1990; Anyukhovsky et al., 1992; Braun and Walsh, 1993; Muntz et al., 1993; LaMorte et al., 1994; Liu et al., 1994; Sah et al., 1996; Hool et al., 1997), but coupling to PTX-sensitive G proteins may occur under some conditions (Steinberg et al., 1985; Han et al., 1989; Del Balzo et al., 1990; Keung and Karliner, 1990; Barrett et al., 1993; Anyukhovsky et al., 1994; Takeda et al., 1994) and has also been suggested in transgenic mice overexpressing WT _1B-adrenoceptors (Akhter et al., 1997a). Moreover, it has recently been reported that ₁-adrenoceptors can also couple to a G protein that is larger than those of the G_q/11 family and designated G_h (Im et al., 1990; Im and Graham, 1990). In later studies, G_h was identified as identical with the enzyme tissue-type transglutaminase II (Nakaoka et al., 1994). The human cardiac ₁-adrenoceptor can also couple to G_h (Hwang et al., 1996). Although the functional consequences of such additional coupling are largely unknown, it is interesting that the extent of ₁-adrenoceptor coupling to G_h can be altered in human heart failure (see Autonomic Responsiveness in Failing Human Heart).

Most studies on the inotropic effects of ₁-adrenoceptor stimulation in human myocardium have used phenylephrine (in the presence of propranolol) as the agonist. As could be expected based on the small receptor number, the inotropic effects elicited by ₁-adrenoceptor stimulation were 15 to 35% of those elicited by -adrenoceptor stimulation (Fig. 2) or receptor-independently by raising extracellular Ca²⁺; such observations were made similarly in atrial (Schümann et al., 1978; Skomedal et al., 1985; Jahnel et al., 1992) and ventricular (Brückner et al., 1984; Aass et al., 1986; Böhm et al., 1988; Jakob et al., 1988; Steinfath et al., 1992b) preparations. However, this may be partly due to the fact that phenylephrine is not a full agonist at any of the three human ₁-adrenoceptor subtypes (Taguchi et al., 1998). Accordingly, it was recently observed that the endogenous transmitter noradrenaline causes a more pronounced inotropic effect in isolated human myocardial strips than phenylephrine (Scholz et al., 1996; Skomedal et al., 1997).

When ₁-adrenoceptor-mediated positive inotropic effects were observed in the human heart, they were accompanied by no change in the action potential configuration (in ventricular myocardium; Jakob et al., 1988) or by a slight decrease in action potential duration (in atrial muscle; Jahnel et al., 1992), although in several animal species, stimulation of myocardial ₁-adrenoceptors prolongs the action potential duration (Brückner et al., 1985; for reviews, see Endoh, 1991; Endoh et al., 1991; Terzic et al., 1993; Li et al., 1997a). Based on these observations, it could be expected that the endogenous catecholamines noradrenaline and adrenaline exert their positive inotropic effects mainly via - rather than ₁-adrenoceptor stimulation. Indeed, two studies with noradrenaline or adrenaline as the agonists were unable to detect inhibition of inotropic effects by the nonselective -adrenoceptor antagonist phentolamine or the selective ₁-adrenoceptor antagonist prazosin (Jakob et al., 1988; Jahnel et al., 1992). In contrast, another study has reported that the contributions of ₁- and -adrenoceptors to the inotropic effects of noradrenaline are similar (Skomedal et al., 1997). Responses to endogenous noradrenaline (released by tyramine administration) were reported to occur via ₁- and -adrenoceptors to 14 and 86%, respectively (Borthne et al., 1995). These divergent results may in part be explained by the use of different tissue sources. Thus, the negative studies have used atrial or ventricular preparations from patients with only mild to moderate heart failure (New York Heart Association functional class II/III), whereas the positive study has used ventricular tissue from patients with severe heart failure (transplant recipients). Because a decrease in -adrenoceptor-mediated positive inotropic effects in advanced heart failure is well documented (Brodde, 1991), a relative enhancement of the -adrenergic effects could be possible under these conditions (but see Autonomic Responsiveness in the Failing Human Heart. A. ₁-Adrenoceptors).

An analysis of the possible contributions of ₁-adrenoceptors to positive inotropic effects of noradrenaline in vivo is difficult because a possible direct effect on the cardiomyocytes may be mimicked and/or concealed by the effects of ₁-adrenoceptor stimulation in the systemic and/or coronary vasculature. One possible experimental approach to this problem is enhancement of endogenous noradrenaline release by tyramine administration. Although i.v. infusion of tyramine caused positive inotropic effects in young healthy volunteers, this was not inhibited by concomitant ₁-adrenoceptor blockade with doxazosin (Schäfers et al., 1997). Another approach is the intracoronary injection or infusion of ₁-adrenoceptor agonists, which at least prevents the problem of afterload elevations. Direct intracoronary infusion of phenylephrine enhanced left ventricular peak (+)dP/dt in a dose-dependent manner; part of this response was sensitive to the -adrenoceptor antagonist phentolamine, indicating that it was mediated by an -adrenoceptor (Landzberg et al., 1991). On the other hand, phentolamine alone did not modify cardiac contractility in that study, indicating that -adrenoceptors do not contribute to the maintenance of basal left ventricular contractile state in humans at rest. Studies with i.v. administration of the ₁-adrenoceptor agonist methoxamine in comparison with angiotensin II have also suggested that a small but detectable component of the inotropic response may occur via ₁-adrenoceptors in humans in vivo (Curiel et al., 1989).

Taken together, these data clearly indicate that stimulation of ₁-adrenoceptors can cause positive inotropic effects in the isolated human heart. Although -adrenoceptor-mediated inotropic effects in rat ventricle appear to occur primarily via _1B-adrenoceptors (Michel et al., 1994b), little is known about the ₁-adrenoceptor subtype causing positive inotropic effects in humans. Although ₁-adrenoceptors may also cause positive inotropic effects in vivo, this may be of limited physiological relevance because the endogenous agonist noradrenaline acts only to a minor degree, if any, via cardiac ₁-adrenoceptors. However, present data are insufficient to exclude that ₁-adrenoceptors participate in the regulation of cardiac force development specifically in settings such as congestive heart failure (see Autonomic Responsiveness in Failing Human Heart) or on blockade of -adrenoceptors. Thus, at least in rat hearts, it has been observed that in vivo treatment with the -adrenoceptor antagonist propranolol increases ₁-adrenoceptor number (Mügge et al., 1985; Steinkraus et al., 1989) and enhances the positive inotropic effects of ₁-adrenoceptor stimulation in vitro (Li et al., 1997b).

Three human ₂-adrenoceptor subtypes exist (_2A, _2B, and _2C; Fig. 1; Bylund et al., 1994); their pharmacological characteristics are shown in Table 2. They are encoded by distinct genes that are located on the human chromosomes 10q23-25, 2, and 4, respectively. The ortholog of the human _2A-adrenoceptor in some species exhibits a markedly different pharmacological recognition profile despite only minor differences in its deduced amino acid sequence and is referred to as the _2D-adrenoceptor (Bylund et al., 1994). Several studies have investigated the presence of ₂-adrenoceptor subtypes in the human heart at the mRNA level using RNase protection assays or RT-PCR. RT-PCR studies have reported the presence of all three subtypes in endocardium at the qualitative level but have failed to detect _2B-adrenoceptor mRNA in left ventricular epicardium despite the presence of the other two subtypes in this tissue (Eason and Liggett, 1993). In contrast, RNase protection assays have not confirmed the presence of _2A-adrenoceptor mRNA in the human heart but have detected mRNA for _2B-adrenoceptors and with an even greater abundance for _2C-adrenoceptors (Perälä et al., 1992; Berkowitz et al., 1994). However, it should be noted that in a direct comparison, even the abundance of mRNA for the _2C-adrenoceptors was 30-fold less than that for the _1A-adrenoceptor (Berkowitz et al., 1994). In the fetal human heart, mRNA for _2A- or _2C-adrenoceptors has not been detected (Perälä et al., 1992). In light of this small ₂-adrenoceptor subtype mRNA abundance, it is not surprising that we have not been successful in demonstrating ₂-adrenoceptors in human heart at the protein level through radioligand binding studies (OEB and MCM, unpublished observations) and are not aware of any other studies to this effect.

View this table: [in this window] [in a new window]   TABLE 2 Pharmacological characterization of 2-adrenoceptor subtypes

Functional studies on human cardiac ₂-adrenoceptors have focused on presynaptic inhibition of noradrenaline release. Thus, ₂-adrenoceptor-mediated prejunctional inhibition has repeatedly been demonstrated in isolated human atrial appendages (Rump et al., 1995a,b; Likungu et al., 1996). The prejunctional inhibitory receptor in the human atrium has originally been classified as an _2C-adrenoceptor (Rump et al., 1995a). This contrasts evidence from a variety of tissues and species, where the prejunctional ₂-adrenoceptor belongs to the _2A subtype or its species ortholog _2D (Trendelenburg et al., 1997). However, this discrepancy between human heart and other tissues should not be overinterpreted for two reasons. First, the pharmacological tools used at the time did not allow a very good discrimination between _2A- and _2C-adrenoceptors. Second, the same authors, using the same tools, have also classified the prejunctional receptor in human kidney as belonging to the _2C subtype (Trendelenburg et al., 1994) but have reclassified it as _2A based on newer tools (Trendelenburg et al., 1997).

The discrepancy between pharmacological classification of prejunctional ₂-adrenoceptors in the human heart as _2A and the apparent absence of corresponding mRNA (see above) is not surprising because the perikarya of the cardiac sympathetic neurons reside in the sympathetic chain.

To test the functional relevance of these in vitro findings for the in vivo setting, studies with intracoronary infusion of the -adrenoceptor antagonist phentolamine have been performed (Parker et al., 1995). In these studies, intracoronary phentolamine infusion did not modify catecholamine spillover in subjects with normal left ventricular function but enhanced it in patients with congestive heart failure. Because a catecholamine release-enhancing effect of -adrenoceptor antagonists depends on the presence of a tonically active catecholamine release and because an enhanced sympathetic drive in congestive heart failure is well documented (Packer, 1992), these data suggest that prejunctional ₂-adrenoceptors play a functional noradrenaline release-inhibiting role in the human heart that becomes evident under conditions of enhanced sympathetic activity.

Three different -adrenoceptor subtypes have been cloned so far and identified pharmacologically: ₁, ₂, and ₃ (Fig. 1; Bylund et al., 1994). These subtypes are encoded by three distinct genes that are located on human chromosomes 10q24-26, 5q31-32, and 8p11-12. The molecular structure of the ₃-adrenoceptor and its gene differ in various ways from those of the ₁- and ₂-adrenoceptors: Thus, the human ₃-adrenoceptor gene has introns, whereas the ₁- and ₂-adrenoceptor genes do not (Granneman et al., 1993; Van Spronsen et al., 1993). Moreover, the human ₃-adrenoceptor lacks sites for phosphorylation by cAMP-dependent protein kinase (PKA) and GRK2 in its carboxyl terminus that are readily found in the ₁- and ₂-adrenoceptors (Hausdorff et al., 1990; Emorine et al., 1991; Strosberg, 1997a). Finally, there are marked species differences between rodent and human ₃-adrenoceptors with respect to expression in white versus brown adipose tissue and the sensitivity to stimulation by certain ₃-adrenoceptor-selective agonists (Strosberg, 1997a), whereas such species differences obviously do not exist for ₁- and ₂-adrenoceptors (Bylund et al., 1994). Recent pharmacological studies, mainly in human and rat cardiac tissue, have proposed the existence of a fourth -adrenoceptor (see below), but this subtype has not been cloned so far. The pharmacological characteristics of -adrenoceptor subtypes are shown in Table 3.

View this table: [in this window] [in a new window]   TABLE 3 Pharmacological characterization of -adrenoceptor subtypes

1. ₁- and ₂-Adrenoceptors. In the human heart, the existence of ₁- and ₂-adrenoceptors has been demonstrated at the mRNA level with RT-PCR and RNase protection assay (Bristow et al., 1993; Ungerer et al., 1993; Engelhardt et al., 1996; Ihl-Vahl et al., 1996), at the protein level with radioligand binding, and in functional studies both in vitro and in vivo (for reviews, see Jones et al., 1989; Bristow et al., 1990; Brodde, 1991; Bristow, 1993; Harding et al., 1994; Brodde et al., 1995a; Kaumann and Molenaar, 1997). The coexistence of ₁- or ₂-adrenoceptors has also been demonstrated on isolated human ventricular cardiomyocytes (Del Monte et al., 1993).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   A, effect of the 1-adrenoceptor antagonist bisoprolol (15 mg p.o. 2 h before agonist infusion) or of propranolol (5 mg i.v. 45 min before agonist infusion) on isoprenaline (left), terbutaline (middle), and adrenaline (right) infusion-induced increases in heart rate in eight healthy male volunteers. Ordinate, increase in heart rate (in  bpm). Abscissa, dose of agonists (in ng/kg/min). Values are means ± S.E. modified from Daul et al. (1995). B, isoprenaline (ISO) and terbutaline (TER) infusion-induced maximal increases in heart rate and maximal shortening of the systolic time intervals (as measure of inotropism) pre-ejection period (PEP) and heart rate-corrected duration of electromechanical systole (QS2) in seven healthy male volunteers. Ordinate, maximal increase in heart rate (left) and shortening of PEP (middle) and QS2 time (right) expressed as maximal percent changes from baseline. Values are means ± S.E. recalculated from Schäfers et al. (1994).

2. Is There a Third (or Fourth) -Adrenoceptor Subtype Present in Human Heart? During the past few years, evidence has accumulated that in addition to ₁- and ₂-adrenoceptors, a third or fourth (or both) -adrenoceptor might exist in the human heart. The existence of a third -adrenoceptor had been originally suggested based on the findings that in guinea pig and cat hearts, "nonconventional" -adrenoceptor antagonists with partial agonistic activity (e.g., pindolol and congeners) exhibited stimulatory properties in concentrations exceeding those required for -adrenoceptor blockade (Kaumann, 1989). The site mediating these effects could be the cloned ₃-adrenoceptor (discovered by Strosberg and his group [Emorine et al., 1989]) and/or "the putative ₄-adrenoceptor" (described by Kaumann, 1996).