**10. Adrenergic receptors**

22 Neuroscience – Dealing with Frontiers

(Heacock and Mahon, 1958, Bindoli et al., 1992, Bindoli et al., 1999). This indole is often represented as a zwitterionic structure in aqueous solutions (Heacock and Mahon, 1958, Remião et al., 2003, Costa et al., 2007) (Figure 5). In summary, the oxidation of catecholamines occurs through two-stages whereby a total of four electrons is removed and

Aminochrome

Aminolutin

N

R\*

R

O N

R

R

R\*

R\*

O

HO

HO

O

O2

O2

HO N

Leucoaminochrome*o*-semiquinone

O2 2-

O2

an indole is formed by cyclization (Costa et al., 2011).

HO

Leucoaminochrome

R

R\*

 **R R\* Adrenaline -OH -CH3 Noradrenaline -OH -H Dopamine -H -H** 

Fig. 5. Postulated pathway for the oxidation of catecholamines. The oxidation process of catecholamines initially involves their conversion to *o*-quinones through o-semiquinones intermediates. The *o*-semiquinone reduces oxygen resulting in superoxide anion (O2●–). The

leucoaminonochrome. The formation of aminochrome from leucoaminochrome is a reaction where a total of two electrons are removed and leucoaminochrome semiquinone is the intermediary with O2●– as a by-product. Aminochrome formed can lead to the formation of

Adrenochrome is the most studied aminochrome due to its stability (Heacock, 1959, Rupp et al., 1994). When adrenochrome is formed and ADR still exists in solution, adrenochrome accelerates the oxidation process of the remaining ADR (Bindoli et al., 1999, Costa et al., 2007). Furthermore, the yield of adrenochrome increases if ADR semi-quinone reacts with O2 to form O2•**–** (Costa et al., 2007). This seems to be a general phenomenon of aminochromes, since it

Once formed, the aminochromes can be transformed into melanins, since they are reactive compounds that easily undergo a series of reactions among them. *In vivo*, this oxidation pathway may be more complex, since other factors such as metal ions or other nucleophilic

*o*-quinone can undergo an irreversible 1, 4-intramolecular cyclization, forming

occurs also with the other catecholamines (Heacock, 1959, Bindoli et al., 1992).

HO N

**HO**

O

HO

O

O

**HO**

**N**

**H R\***

**R**

N

H R\*

R

O2

O2

O2 2-

O2

Catecholamine

aminolutins.

Catecholamine-*o*-semiquinone

N

H R\*

R

Catecholamine-*o*-quinone

#### **10.1 Historic introduction and background**

More than one hundred years ago, Langley proposed, for the first time, the idea of transmitter receptors, by stating "(…) neither the poisons nor the nervous impulse act directly on the contractile substance of the muscle but on some accessory substance. Since this accessory substance is the recipient of stimuli which it transfers to the contractile material, we may speak of it as the receptive substance (…)" (Langley, 1905).

The receptors for NA and ADR are nowadays called adrenoceptors. The adrenoceptors are the cell membrane sites where NA and ADR act. Several discoveries were performed before the concept of adrenoceptors was fully accepted. Dale in 1905 verified that the pressor effect of ADR was reversed by ergotoxine into a depressor effect. Later on, Barger and Dale verified that ADR when injected was able to dilate some vascular beds while constricting others (Barger and Dale, 1910). These facts were ignored until 1948 when Ahlquist performed a series of experiments with several sympathomimetic amines. He concluded that the variation in the pharmacological responses of several sympathomimetic agonists in different organs was related to the different types of receptors involved (Ahlquist, 1948). To the first type of adrenoceptors, he called α and to the second β (Ahlquist, 1948). The scientific contemporary community was not prompt to accept this idea and the theory of two different "sympathins" remained (Cannon and Rosenblueth, 1933). More than ten years later other works confirmed the theory by Ahlquist through the identification of selective antagonists for the two receptor families: phentolamine and ergotamine for αadrenoceptors; dichloroisoprenaline (Powell et al., 1958) and propranolol for βadrenoceptors (Black et al., 1964).

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 25

sensitive pathways such as CaMKII, whilst diacylglycerol activates PKC (Guimarães and Moura, 2001, Westfall and Westfall, 2006). The three cloned α1-adrenoceptor subtypes have significant differences in G protein coupling efficiency (α1A > α1B > α1D) (Docherty, 1998,

Other signalling pathways are also activated by α1-adrenoceptors, namely: stimulation of phospholipase A2 leading to the release of free arachidonate, which is degraded by cyclooxygenase and lipoxygenase to form the bioactive prostaglandins and leukotrienes; Ca2+ influx via protein G; and phospholipase D activation (Docherty, 1998, Zhong and Minneman, 1999, Guimarães and Moura, 2001, Westfall and Westfall, 2006). Some of the responses induced by α1-adrenoceptors are independent of Ca2+ and PKC but involve small G proteins and tyrosine kinases (Zhong and Minneman, 1999). Furthermore, α1 adrenoceptors are able to activate mitogen-activated protein kinase pathways in many cells (Della Rocca et al., 1997). The mitogen-activated protein kinase superfamily, which consists of extracellular signal-regulated kinases 1/2 and three stress-responsive subfamilies, the c-Jun NH2-terminal kinases, p38-mitogen-activated protein kinase (p38-MAPK), and extracellular signal-regulated kinases, is normally stimulated by growth factors and cellular

α2-Adrenoceptors when located presynaptically are responsible for the inhibition of transmitter release (including NA and acetylcholine from autonomic nerves) and are considered modulators of neurotransmission (autoreceptors). The different α2-receptors couple to a variety of effectors (Aantaa et al., 1995, Guimarães and Moura, 2001) and share about 50% in amino acid sequence in important domains (Aantaa et al., 1995). The importance of these receptors was well demonstrated in knockout mice for α2-adrenoceptors (Vieira-Coelho et al., 2009). Brain tissue levels of L-3,4-dihydroxyphenylalanine, dopamine, and NA were significantly higher in the knockout mice for α2A- and α2C-adrenoceptors when compared to wild type. The activity of COMT was higher in all three knockout (α2A-, α2Band α2C-adrenoceptors), while the activities of MAO, dopamine β-hydroxylase or tyrosine

α2-Adrenoceptors are predominantly coupled to the inhibitory G proteins, Gi and G(o), inhibiting the activity of adenylyl cyclase (Rouot et al., 1987, Cotecchia et al., 1990, Surprenant et al., 1992, Aantaa et al., 1995, Wise et al., 1997). The α2-adrenoceptor stimulation leads to the activation of Na+/H+ exchange (Limbird, 1988), inhibition of the opening of voltage-gated Ca2+ channels (Cotecchia et al., 1990) or activation of K+ channels

α2-Adrenoceptors are also found at postjunctional or nonjunctional sites in several tissues, thus their activation may also lead to the activation of other intracellular pathways. In peripheral tissues, postsynaptic α2-receptors are found in vascular and other smooth muscle cells, in adipocytes, and in many types of secretory epithelial cells (intestinal, renal, and endocrine). The activation of α2-adrenoceptors causes platelet aggregation, contraction of vascular smooth muscle, and inhibition of insulin release (Aantaa et al., 1995, Pocock and Richards, 2006, Rang et al., 2007). Intracellular pathways of postjunctional α2-receptors are varied, with activation of phospholipase A2, C, and D, with arachidonic acid mobilization, increase in phosphoinositide hydrolysis, and increase in the intracellular availability of Ca2+ (Limbird, 1988, Cotecchia et al., 1990, MacNulty et al., 1992, Kukkonen et al., 1998). In

Zhong and Minneman, 1999, Guimarães and Moura, 2001).

stress or inflammatory cytokines (Zhang et al., 2005).

hydroxylase were unchanged (Vieira-Coelho et al., 2009).

(Surprenant et al., 1992), all resulting in membrane hyperpolarization.

The development of more selective drugs and the use of molecular cloning technology showed that several adrenoceptor subtypes exist. As Ahlquist stated, two main classes are known, α- and β-adrenoceptors. Each group is further subdivided so that five subtypes are presently recognized: two main α-receptor subtypes (α1 and α2) and three β-receptor subtypes (β1, β2 and β3) (Alexander et al., 2008). The α-receptor subtypes are nowadays accepted to be six subtypes in total (α1A, α1B, α1D, α2A/D, α2B, α2C). Other two adrenoceptors candidates were described (α1L and β4), that may be conformational states of α1A and β1 adrenoceptors, respectively (Guimarães and Moura, 2001, Alexander et al., 2008) .

The pharmacological actions of NA and ADR have been extensively compared *in vivo* and *in vitro*. Both drugs are direct agonists on effectors cells and their actions differ mainly in their ability to stimulate α- and β-receptors. ADR and NA are approximately equipotent in stimulating β1-receptors. NA is a potent α-agonist and has relatively little action on β1 receptors; however, NA is somewhat less potent than ADR on α receptors in most organs (Westfall and Westfall, 2006). The rank order of potency is isoproterenol > ADR > NA for βadrenergic receptors and ADR > NA >> isoproterenol for α-adrenergic receptors (Guimarães, 1975, Westfall and Westfall, 2006, Rang et al., 2007).

In the present review, the classical pharmacological point of view will be approached as it was historically the starting point in the study of the neurotransmission of catecholamines. Although not excluding neurotransmission plasticity and adaptation as key factors for the full understanding of the topic, these issues are still under debate, so they will not be addressed here.

The knowledge of the characteristics of adrenergic receptors and the biochemical and physiological pathways that they regulate increased the understanding of the seemingly contradictory and variable effects of catecholamines on different organs. Although structurally related, different receptors regulate distinct physiological processes by controlling the synthesis or release of a variety of second messengers. The adrenoceptors are coupled to second-messenger systems via G proteins. The responses that follow the activation of adrenergic receptors result from G protein-mediated effects with the generation of second messengers and/or the activation of ion channels (Guimarães and Moura, 2001) (Figure 3).

#### **10.2 Alpha (α)-adrenoceptors**

α1-Adrenoceptors are found in the smooth muscle of the blood vessels, bronchi, gastrointestinal tract, uterus, and bladder. The activation of these receptors is mainly excitatory and results in the contraction of smooth muscle. However, the smooth muscle of the gut wall (but not that of the sphincters) becomes relaxed after activation of α1-receptors. Overall, the α1-adrenoceptors cause vasoconstriction, relaxation of gastrointestinal smooth muscle, salivary secretion, and hepatic glycogenolysis (Pocock and Richards, 2006, Rang et al., 2007). α1-Adrenoceptors are mainly coupled to Gq/11-protein with consequent activation of phospholipase C. Phospholipase C promotes the hydrolysis of phosphatidylinositol bisphosphate producing inositol trisphosphate and diacylglycerol (Docherty, 1998, Zhong and Minneman, 1999, García-Sáinz et al., 2000, Guimarães and Moura, 2001), which act as second messengers. Inositol trisphosphate mediates intracellular Ca2+ release from non mitochondrial pools and, consequently, the activation of other Ca2+ and calmodulin

The development of more selective drugs and the use of molecular cloning technology showed that several adrenoceptor subtypes exist. As Ahlquist stated, two main classes are known, α- and β-adrenoceptors. Each group is further subdivided so that five subtypes are presently recognized: two main α-receptor subtypes (α1 and α2) and three β-receptor subtypes (β1, β2 and β3) (Alexander et al., 2008). The α-receptor subtypes are nowadays accepted to be six subtypes in total (α1A, α1B, α1D, α2A/D, α2B, α2C). Other two adrenoceptors candidates were described (α1L and β4), that may be conformational states of α1A and β1-

The pharmacological actions of NA and ADR have been extensively compared *in vivo* and *in vitro*. Both drugs are direct agonists on effectors cells and their actions differ mainly in their ability to stimulate α- and β-receptors. ADR and NA are approximately equipotent in stimulating β1-receptors. NA is a potent α-agonist and has relatively little action on β1 receptors; however, NA is somewhat less potent than ADR on α receptors in most organs (Westfall and Westfall, 2006). The rank order of potency is isoproterenol > ADR > NA for βadrenergic receptors and ADR > NA >> isoproterenol for α-adrenergic receptors

In the present review, the classical pharmacological point of view will be approached as it was historically the starting point in the study of the neurotransmission of catecholamines. Although not excluding neurotransmission plasticity and adaptation as key factors for the full understanding of the topic, these issues are still under debate, so they will not be

The knowledge of the characteristics of adrenergic receptors and the biochemical and physiological pathways that they regulate increased the understanding of the seemingly contradictory and variable effects of catecholamines on different organs. Although structurally related, different receptors regulate distinct physiological processes by controlling the synthesis or release of a variety of second messengers. The adrenoceptors are coupled to second-messenger systems via G proteins. The responses that follow the activation of adrenergic receptors result from G protein-mediated effects with the generation of second messengers and/or the activation of ion channels (Guimarães and

α1-Adrenoceptors are found in the smooth muscle of the blood vessels, bronchi, gastrointestinal tract, uterus, and bladder. The activation of these receptors is mainly excitatory and results in the contraction of smooth muscle. However, the smooth muscle of the gut wall (but not that of the sphincters) becomes relaxed after activation of α1-receptors. Overall, the α1-adrenoceptors cause vasoconstriction, relaxation of gastrointestinal smooth muscle, salivary secretion, and hepatic glycogenolysis (Pocock and Richards, 2006, Rang et al., 2007). α1-Adrenoceptors are mainly coupled to Gq/11-protein with consequent activation of phospholipase C. Phospholipase C promotes the hydrolysis of phosphatidylinositol bisphosphate producing inositol trisphosphate and diacylglycerol (Docherty, 1998, Zhong and Minneman, 1999, García-Sáinz et al., 2000, Guimarães and Moura, 2001), which act as second messengers. Inositol trisphosphate mediates intracellular Ca2+ release from non mitochondrial pools and, consequently, the activation of other Ca2+ and calmodulin

adrenoceptors, respectively (Guimarães and Moura, 2001, Alexander et al., 2008) .

(Guimarães, 1975, Westfall and Westfall, 2006, Rang et al., 2007).

addressed here.

Moura, 2001) (Figure 3).

**10.2 Alpha (α)-adrenoceptors** 

sensitive pathways such as CaMKII, whilst diacylglycerol activates PKC (Guimarães and Moura, 2001, Westfall and Westfall, 2006). The three cloned α1-adrenoceptor subtypes have significant differences in G protein coupling efficiency (α1A > α1B > α1D) (Docherty, 1998, Zhong and Minneman, 1999, Guimarães and Moura, 2001).

Other signalling pathways are also activated by α1-adrenoceptors, namely: stimulation of phospholipase A2 leading to the release of free arachidonate, which is degraded by cyclooxygenase and lipoxygenase to form the bioactive prostaglandins and leukotrienes; Ca2+ influx via protein G; and phospholipase D activation (Docherty, 1998, Zhong and Minneman, 1999, Guimarães and Moura, 2001, Westfall and Westfall, 2006). Some of the responses induced by α1-adrenoceptors are independent of Ca2+ and PKC but involve small G proteins and tyrosine kinases (Zhong and Minneman, 1999). Furthermore, α1 adrenoceptors are able to activate mitogen-activated protein kinase pathways in many cells (Della Rocca et al., 1997). The mitogen-activated protein kinase superfamily, which consists of extracellular signal-regulated kinases 1/2 and three stress-responsive subfamilies, the c-Jun NH2-terminal kinases, p38-mitogen-activated protein kinase (p38-MAPK), and extracellular signal-regulated kinases, is normally stimulated by growth factors and cellular stress or inflammatory cytokines (Zhang et al., 2005).

α2-Adrenoceptors when located presynaptically are responsible for the inhibition of transmitter release (including NA and acetylcholine from autonomic nerves) and are considered modulators of neurotransmission (autoreceptors). The different α2-receptors couple to a variety of effectors (Aantaa et al., 1995, Guimarães and Moura, 2001) and share about 50% in amino acid sequence in important domains (Aantaa et al., 1995). The importance of these receptors was well demonstrated in knockout mice for α2-adrenoceptors (Vieira-Coelho et al., 2009). Brain tissue levels of L-3,4-dihydroxyphenylalanine, dopamine, and NA were significantly higher in the knockout mice for α2A- and α2C-adrenoceptors when compared to wild type. The activity of COMT was higher in all three knockout (α2A-, α2Band α2C-adrenoceptors), while the activities of MAO, dopamine β-hydroxylase or tyrosine hydroxylase were unchanged (Vieira-Coelho et al., 2009).

α2-Adrenoceptors are predominantly coupled to the inhibitory G proteins, Gi and G(o), inhibiting the activity of adenylyl cyclase (Rouot et al., 1987, Cotecchia et al., 1990, Surprenant et al., 1992, Aantaa et al., 1995, Wise et al., 1997). The α2-adrenoceptor stimulation leads to the activation of Na+/H+ exchange (Limbird, 1988), inhibition of the opening of voltage-gated Ca2+ channels (Cotecchia et al., 1990) or activation of K+ channels (Surprenant et al., 1992), all resulting in membrane hyperpolarization.

α2-Adrenoceptors are also found at postjunctional or nonjunctional sites in several tissues, thus their activation may also lead to the activation of other intracellular pathways. In peripheral tissues, postsynaptic α2-receptors are found in vascular and other smooth muscle cells, in adipocytes, and in many types of secretory epithelial cells (intestinal, renal, and endocrine). The activation of α2-adrenoceptors causes platelet aggregation, contraction of vascular smooth muscle, and inhibition of insulin release (Aantaa et al., 1995, Pocock and Richards, 2006, Rang et al., 2007). Intracellular pathways of postjunctional α2-receptors are varied, with activation of phospholipase A2, C, and D, with arachidonic acid mobilization, increase in phosphoinositide hydrolysis, and increase in the intracellular availability of Ca2+ (Limbird, 1988, Cotecchia et al., 1990, MacNulty et al., 1992, Kukkonen et al., 1998). In

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 27

nasal congestion (Aantaa et al., 1995, Guimarães and Moura, 2001, Westfall and Westfall, 2006, Rang et al., 2007). Inhibition of α1-adrenoceptors has also proven useful to treat hypertension and prostate hypertrophy (Zhong and Minneman, 1999, Westfall and

The α- and β-receptors appear to be located pre- and postsynaptically. The α1- and β1 receptors are mainly in the vicinity of sympathetic adrenergic nerve terminals in peripheral target organs, or distributed in the mammalian brain (Westfall and Westfall, 2006). The α2 and β2-receptors are heterogeneously distributed. Both α2- and β2-receptors may be situated at sites (vascular smooth muscle, platelets and leukocytes) that are relatively remote from nerve terminals and may be activated preferentially by circulating catecholamines (in particular ADR) (Westfall and Westfall, 2006). On the other hand, presynaptically located α2- and β2-adrenoceptors have important roles in the modulation of neurotransmitter release

Responses mediated by adrenoceptors are not static, as they can be modulated and adjusted. The number and function of adrenoceptors on the cell's surface and their elicited responses may be altered by catecholamines themselves, by other hormones and drugs but they can also change with age and disease. The release of neurotransmitters can be modulated by prejunctional autoreceptors. NA or ADR, neuropeptide Y, and ATP (the last two are frequent cotransmitters in the adrenergic transmission) elicit a feedback response on prejunctional receptors (Boehm and Kubista, 2002, Westfall et al., 2002). The most studied autoreceptors have been the prejunctional α2-adrenergic receptors (Aantaa et al., 1995). The α2A- (α2D-, depending on the species investigated) and α2C-adrenergic receptors are the principal prejunctional receptors that inhibit sympathetic neurotransmitter release, whereas the α2Badrenergic receptors may also inhibit the release of transmitters at selected sites (Boehm and Kubista, 2002). Antagonists of these receptors, in turn, can enhance the electrically evoked release of sympathetic neurotransmitters. Neuropeptide Y, acting on Y2 receptors (Chen et al., 1997), and ATP-derived adenosine, acting on P1 (A1) receptors, can also inhibit sympathetic neurotransmitter release, while nucleotides, acting on P2 receptors have inhibitory or facilitator effects, depending on the tissue evaluated (Driessen et al., 1994, Boehm and Kubista, 2002, Hoffmann, 2004). Other heteroreceptors present in sympathetic nerve varicosities can likewise inhibit the release of sympathetic neurotransmitters, namely through the activation of M2 and M4 muscarinic, 5-HT, prostaglandin E2, histamine, enkephalin, and dopamine

receptors (Lefkowitz et al., 1990, Gainetdinov et al., 2004, Hata et al., 2004).

be targets for agonists and antagonists (Boehm and Kubista, 2002).

Enhancement of sympathetic neurotransmitter release can occur after the activation of presynaptic β2-adrenergic, angiotensin II, and nicotinic receptors. All of these receptors can

The modulation of synaptic transmission is a crucial step in homeostasis, where receptors have key roles. The continuous exposure to adrenergic agonists causes a progressive decrease in the capacity to respond to those agents by catecholamine-sensitive cells. This phenomenon, often termed refractoriness, or desensitization, is another "check point" in neuroendrocrine regulation (García-Sáinz et al., 2000, Hoffmann, 2004). Two major types of desensitization have been distinguished: homologous and heterologous. In the homologous, reduced responsiveness is observed exclusively in the receptor originally stimulated. In heterologous desensitization, a decreased responsiveness is observed to an agent or agents

Westfall, 2006).

from sympathetic nerve endings (Figure 3).

addition, α2-adrenoceptors can activate mitogen-activated protein kinases (Della Rocca et al., 1997, Richman and Regan, 1998).

#### **10.3 Beta (β)-adrenoceptors**

The three β-adrenoceptor subtypes are encoded by three different genes located in human chromosomes 10 (β1), 5 (β2), and 8 (β3). Approximately 60% of the amino acid sequence in membrane-spanning domains is the ligand-binding pocket for ADR and NA (Kobilka et al., 1987, Emorine et al., 1989, Guimarães and Moura, 2001).

β1-Adrenoceptors are found in the heart where their activation results in increased rate and force of contraction. They are also present in the sphincter muscle of the gut where their activation leads to relaxation (Pocock and Richards, 2006, Westfall and Westfall, 2006, Rang et al., 2007).

The activation of β2-adrenoceptors in the smooth muscle of certain blood vessels leads to vasodilatation. They are also present in the bronchial smooth muscle where they mediate bronchodilatation. Relaxation of visceral smooth muscle, hepatic glycogenolysis, and muscle tremor occur after β2-adrenoceptors activation. Postsynaptic β2-receptors can be found in the myocardium, as well as on vascular and some smooth muscle cells (Gauthier et al., 1996, Gauthier et al., 2000, Pocock and Richards, 2006, Rang et al., 2007). The stimulation of prejunctional β2-adrenoceptors leads to NA release in neurons (Boehm and Kubista, 2002).

β3-Adrenoceptors are present in adipose tissue, where they initiate lipolysis in white adipose tissue (Pocock and Richards, 2006, Rang et al., 2007). They are involved in the thermogenesis process that takes place in brown adipose tissue (Gauthier et al., 1996). β3- Adrenoceptors are also present in the heart although their functions are not fully understood (Gauthier et al., 1996, Gauthier et al., 2000).

All β-receptor subtypes (β1, β2, and β3) are coupled to the stimulatory G protein (Gs) leading to the activation of adenylyl cyclase and accumulation of the second messenger, cyclic adenosine monophosphate (cAMP) (Frielle et al., 1987, Emorine et al., 1989, Brown, 1990, Westfall and Westfall, 2006). Accumulation of cAMP leads to PKA activation with phosphorylation of several proteins, whose functions are changed as a result.

Curiously, several works indicate that, under certain circumstances, β-adrenoceptors can couple to Gi in addition to Gs. In fact, β3-receptors interact with both Gs and Gi, whereas β1 receptors couple predominantly to Gs (Asano et al., 1984, Chaudhry et al., 1994, Gauthier et al., 1996). β2-Receptors usually bind to Gs but, after sustained activation, they couple to Gi (Xiao et al., 1995, Lefkowitz et al., 2002).

#### **10.4 Location and regulation of adrenoceptors**

The adrenoceptors are targets for many important drugs in therapeutics, including those used for treatment of cardiovascular diseases, asthma, prostatic hypertrophy, nasal congestion, obesity, and pain (Guimarães and Moura, 2001, Pocock and Richards, 2006). From a therapeutic standpoint, there are many occasions where the β-adrenoceptor selective stimulation (asthma, atrioventricular block, obesity) or blockage (hypertension, coronary insufficiency) is desired. α2-Agonists are used in hypertension and α1-agonist in

addition, α2-adrenoceptors can activate mitogen-activated protein kinases (Della Rocca et al.,

The three β-adrenoceptor subtypes are encoded by three different genes located in human chromosomes 10 (β1), 5 (β2), and 8 (β3). Approximately 60% of the amino acid sequence in membrane-spanning domains is the ligand-binding pocket for ADR and NA (Kobilka et al.,

β1-Adrenoceptors are found in the heart where their activation results in increased rate and force of contraction. They are also present in the sphincter muscle of the gut where their activation leads to relaxation (Pocock and Richards, 2006, Westfall and Westfall, 2006, Rang

The activation of β2-adrenoceptors in the smooth muscle of certain blood vessels leads to vasodilatation. They are also present in the bronchial smooth muscle where they mediate bronchodilatation. Relaxation of visceral smooth muscle, hepatic glycogenolysis, and muscle tremor occur after β2-adrenoceptors activation. Postsynaptic β2-receptors can be found in the myocardium, as well as on vascular and some smooth muscle cells (Gauthier et al., 1996, Gauthier et al., 2000, Pocock and Richards, 2006, Rang et al., 2007). The stimulation of prejunctional β2-adrenoceptors leads to NA release in neurons (Boehm and Kubista, 2002).

β3-Adrenoceptors are present in adipose tissue, where they initiate lipolysis in white adipose tissue (Pocock and Richards, 2006, Rang et al., 2007). They are involved in the thermogenesis process that takes place in brown adipose tissue (Gauthier et al., 1996). β3- Adrenoceptors are also present in the heart although their functions are not fully

All β-receptor subtypes (β1, β2, and β3) are coupled to the stimulatory G protein (Gs) leading to the activation of adenylyl cyclase and accumulation of the second messenger, cyclic adenosine monophosphate (cAMP) (Frielle et al., 1987, Emorine et al., 1989, Brown, 1990, Westfall and Westfall, 2006). Accumulation of cAMP leads to PKA activation with

Curiously, several works indicate that, under certain circumstances, β-adrenoceptors can couple to Gi in addition to Gs. In fact, β3-receptors interact with both Gs and Gi, whereas β1 receptors couple predominantly to Gs (Asano et al., 1984, Chaudhry et al., 1994, Gauthier et al., 1996). β2-Receptors usually bind to Gs but, after sustained activation, they couple to Gi

The adrenoceptors are targets for many important drugs in therapeutics, including those used for treatment of cardiovascular diseases, asthma, prostatic hypertrophy, nasal congestion, obesity, and pain (Guimarães and Moura, 2001, Pocock and Richards, 2006). From a therapeutic standpoint, there are many occasions where the β-adrenoceptor selective stimulation (asthma, atrioventricular block, obesity) or blockage (hypertension, coronary insufficiency) is desired. α2-Agonists are used in hypertension and α1-agonist in

phosphorylation of several proteins, whose functions are changed as a result.

1997, Richman and Regan, 1998).

1987, Emorine et al., 1989, Guimarães and Moura, 2001).

understood (Gauthier et al., 1996, Gauthier et al., 2000).

(Xiao et al., 1995, Lefkowitz et al., 2002).

**10.4 Location and regulation of adrenoceptors** 

**10.3 Beta (β)-adrenoceptors** 

et al., 2007).

nasal congestion (Aantaa et al., 1995, Guimarães and Moura, 2001, Westfall and Westfall, 2006, Rang et al., 2007). Inhibition of α1-adrenoceptors has also proven useful to treat hypertension and prostate hypertrophy (Zhong and Minneman, 1999, Westfall and Westfall, 2006).

The α- and β-receptors appear to be located pre- and postsynaptically. The α1- and β1 receptors are mainly in the vicinity of sympathetic adrenergic nerve terminals in peripheral target organs, or distributed in the mammalian brain (Westfall and Westfall, 2006). The α2 and β2-receptors are heterogeneously distributed. Both α2- and β2-receptors may be situated at sites (vascular smooth muscle, platelets and leukocytes) that are relatively remote from nerve terminals and may be activated preferentially by circulating catecholamines (in particular ADR) (Westfall and Westfall, 2006). On the other hand, presynaptically located α2- and β2-adrenoceptors have important roles in the modulation of neurotransmitter release from sympathetic nerve endings (Figure 3).

Responses mediated by adrenoceptors are not static, as they can be modulated and adjusted. The number and function of adrenoceptors on the cell's surface and their elicited responses may be altered by catecholamines themselves, by other hormones and drugs but they can also change with age and disease. The release of neurotransmitters can be modulated by prejunctional autoreceptors. NA or ADR, neuropeptide Y, and ATP (the last two are frequent cotransmitters in the adrenergic transmission) elicit a feedback response on prejunctional receptors (Boehm and Kubista, 2002, Westfall et al., 2002). The most studied autoreceptors have been the prejunctional α2-adrenergic receptors (Aantaa et al., 1995). The α2A- (α2D-, depending on the species investigated) and α2C-adrenergic receptors are the principal prejunctional receptors that inhibit sympathetic neurotransmitter release, whereas the α2Badrenergic receptors may also inhibit the release of transmitters at selected sites (Boehm and Kubista, 2002). Antagonists of these receptors, in turn, can enhance the electrically evoked release of sympathetic neurotransmitters. Neuropeptide Y, acting on Y2 receptors (Chen et al., 1997), and ATP-derived adenosine, acting on P1 (A1) receptors, can also inhibit sympathetic neurotransmitter release, while nucleotides, acting on P2 receptors have inhibitory or facilitator effects, depending on the tissue evaluated (Driessen et al., 1994, Boehm and Kubista, 2002, Hoffmann, 2004). Other heteroreceptors present in sympathetic nerve varicosities can likewise inhibit the release of sympathetic neurotransmitters, namely through the activation of M2 and M4 muscarinic, 5-HT, prostaglandin E2, histamine, enkephalin, and dopamine receptors (Lefkowitz et al., 1990, Gainetdinov et al., 2004, Hata et al., 2004).

Enhancement of sympathetic neurotransmitter release can occur after the activation of presynaptic β2-adrenergic, angiotensin II, and nicotinic receptors. All of these receptors can be targets for agonists and antagonists (Boehm and Kubista, 2002).

The modulation of synaptic transmission is a crucial step in homeostasis, where receptors have key roles. The continuous exposure to adrenergic agonists causes a progressive decrease in the capacity to respond to those agents by catecholamine-sensitive cells. This phenomenon, often termed refractoriness, or desensitization, is another "check point" in neuroendrocrine regulation (García-Sáinz et al., 2000, Hoffmann, 2004). Two major types of desensitization have been distinguished: homologous and heterologous. In the homologous, reduced responsiveness is observed exclusively in the receptor originally stimulated. In heterologous desensitization, a decreased responsiveness is observed to an agent or agents

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 29

well known. It is absolutely certain that, as it happened during the twentieth century, the new century will bring new and fascinating discoveries in this area that will challenge existing preconceptions. However, the knowledge gathered until this point resulted of the work of several notorious scientists whose work should not be forgotten but remembered

Adrenaline; Epinephrine (ADR); Adenosine-5'-triphosphate (ATP); Adenosine triphosphatase (ATPase); Calcium ion (Ca2+); Ca2+/calmodulin-dependent protein kinase II (CaMKII); Cyclic adenosine monophosphate (cAMP); Catechol-*O*-methyltransferase (COMT); Dihydroxyphenylglycol (DHPG); Deoxyribonucleic acid (DNA); Inhibitory G protein (Gi); Stimulatory G protein (Gs); 5-Hydroxytriptamine; Serotonin (5-HT); Homovanillic acid (HVA); Potassium ion (K+); Half-saturation constant; Michaelis constant (Km); Monoamine oxidase (MAO); Magnesium ion (Mg2+); Sodium ion (Na+); Noradrenaline; Norepinephrine (NA); Noradrenaline transporter (NET); Superoxide anion (O2●–); Organic cation transporters (OCT); Hydroxyl radical (HO●); Phenylethanolamine Nmethyltransferase; Noradrenaline N-methyltransferase (PMNT); Ribonucleic acid (RNA); Vanillylmandelic acid; Methoxy-4-hydroxymandelic acid (VMA); Vesicular monoamine

This work received financial support from the Portuguese State through "Fundação para a Ciência e Tecnologia" (FCT) (project PPCDT/SAU-OBS/55849/2004 & POCI/SAU-OBS/55849/2004). Vera M. Costa acknowledges FCT for her Pos-Doc grant (SFRH/BPD/63746/2009). The authors acknowledge Joana Macedo for her technical

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and valued.

**11. Abbreviations** 

transporter (VMAT).

**13. References** 

49:78-85.

**12. Acknowledgments** 

assistance with figure 3 included in the manuscript.

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unrelated to the initial stimulus. Certainly, this classification is only operational and both types of desensitization may occur simultaneously in the cells (García-Sáinz et al., 2000). Even so, the mechanisms are equally complex and need further characterization.

β-Adrenoceptor feedback regulation is common; however, β-receptors differ in the extent to which they can undergo such regulation, with the β2-receptors being the most susceptible. Upon challenge with an agonist, the β2-receptor couples to Gs and activates adenylyl cyclase to form cAMP. cAMP leads to stimulation of cyclic AMP-dependent protein kinases, like PKA, with consequent phosphorylation of β-receptor. That phosphorylation provides the signal for β-arrestin recruitment. Arrestins constitute a large family of widely expressed proteins (Baillie and Houslay, 2005, Westfall and Westfall, 2006). β-arrestin translocation from the cytosol to the activated β-receptor physically blocks the interaction of the receptor with its cellular effectors, presumably due to steric hindrance (Lefkowitz and Shenoy, 2005). The receptor phosphorylation followed by β-arrestin binding has been linked to subsequent endocytosis of the receptor. The capacity of β-arrestins to bind to the structural protein clathrin, initiating the internalization of phosphorylated receptors into vesicles, facilitates endocytosis (Goodman et al., 1996, Nelson et al., 2008). In addition to blunting responses that require the presence of the receptor on the surface of the cell, these regulatory processes may also contribute to novel mechanisms of receptor signalling via intracellular pathways (Baillie and Houslay, 2005).

Receptor desensitization may also be mediated by second messenger feedback. For example, β-adrenoceptor-mediated cAMP accumulation leads to activation of PKA, which can phosphorylate residues of β-receptors, which results in their own inhibition. For the β2 receptor, phosphorylation occurs on serine residues both in the third cytoplasmic loop and in the carboxyl terminal tail of the receptor. β3-Receptors do not suffer down regulation, since they lack recognition sites for the cAMP dependent kinases activated by stimulation of β-adrenoceptors (Gauthier et al., 1996).

Although less addressed, α-receptors desensitization has gained increased interest (García-Sáinz et al., 2000). Current data indicate that the decrease in receptor activity is associated with a homologous desensitization mechanism. The activation of PKC by Gq-coupled receptors may lead to phosphorylation of this class of G protein-coupled receptors. That phosphorylation constitutes a very substantial sterical impediment for the effective interaction of receptors with G proteins. Receptor phosphorylation is associated with receptor internalization and β-arrestins are involved (García- Sáinz et al., 2000).

In the case of α-receptors heterologous desensitization, it is not completely clear whether kinases, arrestins, or other molecules play the main role (García-Sáinz et al., 2000). However, activated PKA or PKC may phosphorylate any structurally similar receptor with the appropriate consensus sites for phosphorylation, a process which is considered a heterologous desensitization (Gainetdinov et al., 2004, Hoffmann, 2004).11. Summary

The autonomic nervous system is considered responsible for organ specific adjustments to the environment, while the endocrine system regulates more generalized adaptations by releasing hormones into the systemic circulation that act on "distant places" (Westfall and Westfall, 2006). NA and ADR play crucial roles in the interaction between the autonomic and endocrine systems when the body has to adjust to several and varied situations. It is of outmost importance that their roles and all the participants in the "adrenergic" system are well known. It is absolutely certain that, as it happened during the twentieth century, the new century will bring new and fascinating discoveries in this area that will challenge existing preconceptions. However, the knowledge gathered until this point resulted of the work of several notorious scientists whose work should not be forgotten but remembered and valued.
