**1. Introduction**

### **1.1 An overview on the role of catecholamines: Basic physiology and pharmacology**

Tremendous advances in the knowledge of catecholamines occurred in the last century, namely the discovery of their chemical structure, the notion of adrenoceptors by Ahlquist or the characterization of (at least in part) their transduction pathways. General concepts related to catecholamines, namely their synthesis, storage, reuptake, release, action termination, the adrenergic receptors, and their transduction pathways will be approached in this chapter. Additionally, the levels of catecholamines and metabolites found in human plasma and urine will be briefly addressed. Special focus will be given to adrenaline (ADR) and noradrenaline (NA) since they are the main actors of the human adaptation to the environment. ADR and NA are responsible for the cross talk between the sympathetic nervous system (and central nervous system) and adrenal medulla.

#### **2. Historic introduction and background**

In 1856, Vulpian applied a solution of ferric chloride to slices of adrenal glands and noticed that the medulla stained green while the cortex did not. He also observed that the same reaction occurred in samples of venous blood leaving the adrenal, but not in the arterial blood entering the gland. To account for these observations, Vulpian assumed that the medulla synthesized a substance that was released into the circulation (Vulpian, 1856). In 1895, Oliver and Schäfer demonstrated the first pharmacological action of catecholamines when they showed that the administration of extracts of adrenal gland led to the rise of arterial blood pressure (Oliver and Schäfer, 1895).

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 3

adaptation to a stressful situation. The main function of NA is to orchestrate the response of the body to stress culminating in increased heart rate and blood pressure, and enhanced energy mobilization and neural reflexes (Wang et al., 1999, Westfall and Westfall, 2006).

NA is a neurotransmitter found in the peripheral and central nervous system, where it regulates a numerous assortment of physiological processes that include mood, arousal, learning and memory, blood flow, and metabolism (Axelrod and Kopin, 1969, Xu et al., 2000, Nolte, 2009). As a central nervous system neurotransmitter, NA is synthesized primarily in the brainstem *nuclei locus ceruleus* and *subceruleus*, which project rostrally to virtually every region of the midbrain and forebrain, dorsoventrally to the cerebellum, and caudally to lumbar segments of the spinal cord (Wang et al., 1999, Westfall and Westfall, 2006, Nolte, 2009). Additionally, the A1, C1, A2, C2, C3, A4, A5, and A7 noradrenergic cell groups also provide projections into the brain. Noradrenergic projections are identified in virtually every region of the midbrain, forebrain and cerebellum (Nolte, 2009). In the peripheral nervous system, NA is synthesized and released from sympathetic neurons connected to a wide variety of endocrine organs and other tissues (Axelrod and Kopin, 1969, Wang et al., 1999, Nolte, 2009). This extensive projection pattern throughout the neuroaxis defines NA as a general regulator of neurotransmission. NA is directly involved in mood stabilization, sleep regulation, aggression and in the general degree of alertness and arousal. NA is also involved in the central control over the endocrine and autonomic nervous

Although generally treated only as an endocrine hormone, ADR also acts as a neurotransmitter released in central and peripheral adrenergic neurons (Loewi, 1936, Jarrott, 1970, Fuller, 1982). Indeed, Loewi demonstrated the presence of an "acceleranstoff" released from sympathetic neurons that innervated the heart of frogs (Loewi, 1921), later found to be ADR (Loewi, 1936, Azuma et al., 1965, Norberg and McIsaac, 1967). The presence of ADR in the mammalian central nervous system was recognized both in biochemical and neuroanatomical studies (Lew et al., 1977, Pendleton et al., 1978, Moore and Bloom, 1979, Armstrong et al., 1982, Goodchild et al., 1984). The existence and function of only ADRsynthesizing neurons are not without controversy (Mefford, 1987). The confirmation of specific neurons in the central nervous system that contain ADR occurred after the development of sensitive enzymatic assays and immunocytochemical staining techniques for phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for the synthesis of ADR (Axelrod, 1962, Lew et al., 1977, Pendleton et al., 1978). Subsequently, neuroanatomical studies using antibodies against PNMT showed the presence of ADRsynthesizing neurons in the C1, C2, and C3 cell groups of the *medulla oblongata* and the *nucleus tractus solitarii* in multiple species (Goodchild et al., 1984, Carlton et al., 1987, Carlton et al., 1991), including humans (Burke et al., 1986, Halliday et al., 1988, Kitahama et al., 1988). Many other studies have shown the existence of adrenergic neurons in the medullar reticular formation that make restricted connections to a few pontine and diencephalic *nuclei*, eventually coursing as far rostrally as the paraventricular nucleus of the dorsal midline thalamus (Armstrong et al., 1982, Chamba and Renaud, 1983, Goodchild et al., 1984, Ross et al., 1984b, Stolk et al., 1984, Bloom, 2006). It is recognized nowadays that ADR synthesizing neurons are involved in cardiovascular homeostasis in both physiological and pathophysiological conditions (Moore and Bloom, 1979, Goodchild et al., 1984, Reis et al.,

systems (Wang et al., 1999, Westfall and Westfall, 2006, Nolte, 2009).

1984, Ross et al., 1984a, Johansson et al., 1997).

In 1897, Abel and Crawford obtained a compound that they called "epinephrin" (Abel and Crawford, 1897). The mono-benzoyl derivative isolated was however less active than the crude extracts, so the quest for the bioactive compound continued. In 1901, Takamine, an industrial chemist, isolated the active principle, then named "adrenalin" (Takamine, 1902) (Figure 1). Takamine's notation was adopted by the Europeans but not by North American researchers, thus resulting in the different nomenclature of both sides of the Atlantic Ocean.

Fig. 1. Chemical structures of biogenic catecholamines.

Catecholamines soon became the centre of one of the most endurable and passionate scientific discussions, with tremendous breakthroughs that crossed the twentieth century. When reporting to catecholamines, under the general heading are NA (also known as norepinephrine), the principal transmitter of sympathetic postganglionic fibres and certain tracts of the central nervous system; dopamine, a known transmitter of the mammalian nigrostriatal, mesocortical, and mesolimbic neuronal pathways; and ADR (also known as epinephrine), the major hormone of the adrenal medulla. Collectively, these three amines are called biogenic catecholamines (Westfall and Westfall, 2006) and are involved in the regulation of motor coordination, learning and memory, sleep-wake cycle regulation, as well as in endocrine and visceral functions (Wevers et al., 1999).

#### **3. Structure and main location of catecholamine-releasing sites**

NA, ADR, and dopamine are known as catecholamines, as they contain a catechol moiety, an amine side-chain and they are all derived from the amino acid tyrosine (Westfall and Westfall, 2006, Baynes and Dominiczak, 2007, Rang et al., 2007) (Figure 1). Catecholamines share with 5-hydroxytryptamine (5-HT; serotonin) the amino side chain and altogether are known as biogenic amines. Similar compounds, like isoproterenol (previously isoprenaline), a synthetic derivative of NA, are catecholamines but not biogenic amines (Rang et al., 2007).

Although many before postulated it, von Euler was the first to show that NA was the main neurotransmitter in the sympathetic nervous system (von Euler, 1946). Sympathetic nerves arise from the spinal cord and run to the ganglia situated close to it; from those ganglia the postganglionic noradrenergic nerves run to the target tissues. Stimulation of the sympathetic nerves is inseparable from the "fight or flight" response, which results in the secretion of corticosteroids by the adrenal cortex and the release of ADR and NA by the adrenal medulla and sympathetic nerves (Wang et al., 1999, Baynes and Dominiczak, 2007). Together with ADR and corticosteroids, NA helps to coordinate different body part responses for

In 1897, Abel and Crawford obtained a compound that they called "epinephrin" (Abel and Crawford, 1897). The mono-benzoyl derivative isolated was however less active than the crude extracts, so the quest for the bioactive compound continued. In 1901, Takamine, an industrial chemist, isolated the active principle, then named "adrenalin" (Takamine, 1902) (Figure 1). Takamine's notation was adopted by the Europeans but not by North American researchers, thus resulting in the different nomenclature of both sides of the Atlantic Ocean.

Catecholamines soon became the centre of one of the most endurable and passionate scientific discussions, with tremendous breakthroughs that crossed the twentieth century. When reporting to catecholamines, under the general heading are NA (also known as norepinephrine), the principal transmitter of sympathetic postganglionic fibres and certain tracts of the central nervous system; dopamine, a known transmitter of the mammalian nigrostriatal, mesocortical, and mesolimbic neuronal pathways; and ADR (also known as epinephrine), the major hormone of the adrenal medulla. Collectively, these three amines are called biogenic catecholamines (Westfall and Westfall, 2006) and are involved in the regulation of motor coordination, learning and memory, sleep-wake cycle regulation, as

NA, ADR, and dopamine are known as catecholamines, as they contain a catechol moiety, an amine side-chain and they are all derived from the amino acid tyrosine (Westfall and Westfall, 2006, Baynes and Dominiczak, 2007, Rang et al., 2007) (Figure 1). Catecholamines share with 5-hydroxytryptamine (5-HT; serotonin) the amino side chain and altogether are known as biogenic amines. Similar compounds, like isoproterenol (previously isoprenaline), a synthetic derivative of NA, are catecholamines but not biogenic amines (Rang et al.,

Although many before postulated it, von Euler was the first to show that NA was the main neurotransmitter in the sympathetic nervous system (von Euler, 1946). Sympathetic nerves arise from the spinal cord and run to the ganglia situated close to it; from those ganglia the postganglionic noradrenergic nerves run to the target tissues. Stimulation of the sympathetic nerves is inseparable from the "fight or flight" response, which results in the secretion of corticosteroids by the adrenal cortex and the release of ADR and NA by the adrenal medulla and sympathetic nerves (Wang et al., 1999, Baynes and Dominiczak, 2007). Together with ADR and corticosteroids, NA helps to coordinate different body part responses for

Fig. 1. Chemical structures of biogenic catecholamines.

well as in endocrine and visceral functions (Wevers et al., 1999).

2007).

**3. Structure and main location of catecholamine-releasing sites**

adaptation to a stressful situation. The main function of NA is to orchestrate the response of the body to stress culminating in increased heart rate and blood pressure, and enhanced energy mobilization and neural reflexes (Wang et al., 1999, Westfall and Westfall, 2006).

NA is a neurotransmitter found in the peripheral and central nervous system, where it regulates a numerous assortment of physiological processes that include mood, arousal, learning and memory, blood flow, and metabolism (Axelrod and Kopin, 1969, Xu et al., 2000, Nolte, 2009). As a central nervous system neurotransmitter, NA is synthesized primarily in the brainstem *nuclei locus ceruleus* and *subceruleus*, which project rostrally to virtually every region of the midbrain and forebrain, dorsoventrally to the cerebellum, and caudally to lumbar segments of the spinal cord (Wang et al., 1999, Westfall and Westfall, 2006, Nolte, 2009). Additionally, the A1, C1, A2, C2, C3, A4, A5, and A7 noradrenergic cell groups also provide projections into the brain. Noradrenergic projections are identified in virtually every region of the midbrain, forebrain and cerebellum (Nolte, 2009). In the peripheral nervous system, NA is synthesized and released from sympathetic neurons connected to a wide variety of endocrine organs and other tissues (Axelrod and Kopin, 1969, Wang et al., 1999, Nolte, 2009). This extensive projection pattern throughout the neuroaxis defines NA as a general regulator of neurotransmission. NA is directly involved in mood stabilization, sleep regulation, aggression and in the general degree of alertness and arousal. NA is also involved in the central control over the endocrine and autonomic nervous systems (Wang et al., 1999, Westfall and Westfall, 2006, Nolte, 2009).

Although generally treated only as an endocrine hormone, ADR also acts as a neurotransmitter released in central and peripheral adrenergic neurons (Loewi, 1936, Jarrott, 1970, Fuller, 1982). Indeed, Loewi demonstrated the presence of an "acceleranstoff" released from sympathetic neurons that innervated the heart of frogs (Loewi, 1921), later found to be ADR (Loewi, 1936, Azuma et al., 1965, Norberg and McIsaac, 1967). The presence of ADR in the mammalian central nervous system was recognized both in biochemical and neuroanatomical studies (Lew et al., 1977, Pendleton et al., 1978, Moore and Bloom, 1979, Armstrong et al., 1982, Goodchild et al., 1984). The existence and function of only ADRsynthesizing neurons are not without controversy (Mefford, 1987). The confirmation of specific neurons in the central nervous system that contain ADR occurred after the development of sensitive enzymatic assays and immunocytochemical staining techniques for phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for the synthesis of ADR (Axelrod, 1962, Lew et al., 1977, Pendleton et al., 1978). Subsequently, neuroanatomical studies using antibodies against PNMT showed the presence of ADRsynthesizing neurons in the C1, C2, and C3 cell groups of the *medulla oblongata* and the *nucleus tractus solitarii* in multiple species (Goodchild et al., 1984, Carlton et al., 1987, Carlton et al., 1991), including humans (Burke et al., 1986, Halliday et al., 1988, Kitahama et al., 1988). Many other studies have shown the existence of adrenergic neurons in the medullar reticular formation that make restricted connections to a few pontine and diencephalic *nuclei*, eventually coursing as far rostrally as the paraventricular nucleus of the dorsal midline thalamus (Armstrong et al., 1982, Chamba and Renaud, 1983, Goodchild et al., 1984, Ross et al., 1984b, Stolk et al., 1984, Bloom, 2006). It is recognized nowadays that ADR synthesizing neurons are involved in cardiovascular homeostasis in both physiological and pathophysiological conditions (Moore and Bloom, 1979, Goodchild et al., 1984, Reis et al., 1984, Ross et al., 1984a, Johansson et al., 1997).

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 5

NA, studies on distinct central nervous system regions revealed that the distribution of dopamine and NA neurons is markedly different (Bloom, 2006). More than half of the central nervous system catecholamine content is dopamine and extremely large amounts are found in the basal ganglia (especially the *caudate nucleus*), the *nucleus accumbens*, the olfactory tubercle, the central nucleus of the amygdala, the median eminence, and restricted fields of the frontal cortex (Bloom, 2006). Of the wide variety of connections, the greatest attention has been given to the long projections between the major dopaminecontaining nuclei located at the substantia nigra and ventral tegmentum and their targets in the striatum. In addition, two important central dopaminergic systems are functionally located outside the blood-brain barrier: in the chemoceptor trigger zone and in the anterior pituitary. In the periphery, dopamine is synthesized in epithelial cells of the proximal tubules and it is thought to exert local diuretic and natriuretic effects in the kidneys (Esler et al., 1990, Bloom, 2006). Likewise, dopamine receptors are found in the proximal gastrointestinal tract where their activation delays gastric emptying. Dopaminergic receptors are also present in renal, mesenteric, coronary and intracerebral arteries (Bloom, 2006). At the cellular level, the actions of dopamine depend on the expression of the different receptor subtypes and the contingent actions of other transmitters to the same target neurons (Bloom, 2006). This catecholamine will not be deeply addressed in the present work, except in specific circumstances where it can be of

The mechanisms of catecholamine synthesis, storage, release, and binding have been mainly studied in sympathetically innervated organs and in the adrenal medulla (Westfall and

The steps that occur in the synthesis of dopamine, NA, and ADR are shown in Figure 2. In the cytoplasm, tyrosine is sequentially 3-hydroxylated by tyrosine hydroxylase to form L-3,4-dihydroxyphenylalanine, which is then decarboxylated to form dopamine by L-3,4 dihydroxyphenylalanine decarboxylase (also known as aromatic L-amino acid decarboxylase) (Axelrod, 1962). Dopamine β-hydroxylase β-hydroxylates dopamine to yield NA, which is N-methylated by PNMT to form ADR, in adrenergic neurons and other ADR producing cells (Rang et al., 2007). The enzymes involved in this synthesis have been

In adrenergic neurons, the enzymes that participate in NA formation are synthesized in the cell bodies of neurons and are then transported along the axon to the terminals. In the course of NA synthesis, both the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine and the decarboxylation of L-3,4-dihydroxyphenylalanine to dopamine take place in the cytoplasm. About half of the dopamine formed in the cytoplasm is then actively transported into the dopamine β-hydroxylase-containing storage vesicles, where it is converted to NA. The remainder escapes active transport to the vesicles and is deaminated to 3,4 dihydroxyphenylacetic acid and subsequently *O*-methylated to homovanillic acid (HVA) (Westfall and Westfall, 2006). In ADR-producing cells, the NA formed in the vesicles leaves them and it is methylated in the cytoplasm by PNMT to yield ADR. ADR then re-enters the

relevance to the theme.

Westfall, 2006).

**4. Synthesis of catecholamines** 

identified, cloned, and characterized (Nagatsu, 1991).

In adults, ADR accounts for approximately 80% of adrenal medulla catecholamine content, with NA making up most of the remaining (von Euler, 1972). Although the adrenal gland and neurons are the most prominent sites of PNMT expression in the adult mammal, this enzyme has also been detected in the retina (Hadjiconstantinou et al., 1983, Foster et al., 1985, Park et al., 1986), spleen (Pendleton et al., 1978), lungs (Pendleton et al., 1978, Kennedy et al., 1990), and heart (Axelrod, 1962, Pendleton et al., 1978, Culman et al., 1987, Torda et al., 1987, Kennedy and Ziegler, 1991). ADR triggers coordinated metabolic and physiological processes in response to stress (Westfall and Westfall, 2006). ADR is more active than NA in the heart and lungs, it causes redirection of blood from the skin to skeletal muscle and it has important stimulatory effects in the glycogen metabolism of the liver (Westfall and Westfall, 2006, Baynes and Dominiczak, 2007, Rang et al., 2007).

Dopamine (3,4-dihydroxyphenylethylamine) is the immediate metabolic precursor of NA and ADR (Figure 2). Although dopamine originally was regarded only as a precursor of

Fig. 2. The synthesis of catecholamines. Tyrosine hydroxylase hydroxylates tyrosine and forms L-3,4-dihydroxyphenylalanine (L-dopa). L-dopa is decarboxylated to form dopamine through the catalysis of dopa decarboxylase. Dopamine β-hydroxylase β-hydroxylates dopamine to yield noradrenaline. Noradrenaline is N-methylated by phenylethanolamine N-methyltransferase to form adrenaline in adrenergic neurons and other adrenaline producing cells.

In adults, ADR accounts for approximately 80% of adrenal medulla catecholamine content, with NA making up most of the remaining (von Euler, 1972). Although the adrenal gland and neurons are the most prominent sites of PNMT expression in the adult mammal, this enzyme has also been detected in the retina (Hadjiconstantinou et al., 1983, Foster et al., 1985, Park et al., 1986), spleen (Pendleton et al., 1978), lungs (Pendleton et al., 1978, Kennedy et al., 1990), and heart (Axelrod, 1962, Pendleton et al., 1978, Culman et al., 1987, Torda et al., 1987, Kennedy and Ziegler, 1991). ADR triggers coordinated metabolic and physiological processes in response to stress (Westfall and Westfall, 2006). ADR is more active than NA in the heart and lungs, it causes redirection of blood from the skin to skeletal muscle and it has important stimulatory effects in the glycogen metabolism of the liver (Westfall and Westfall,

Dopamine (3,4-dihydroxyphenylethylamine) is the immediate metabolic precursor of NA and ADR (Figure 2). Although dopamine originally was regarded only as a precursor of

HO

HO

HO

*Phenyletanolamine* 

Fig. 2. The synthesis of catecholamines. Tyrosine hydroxylase hydroxylates tyrosine and forms L-3,4-dihydroxyphenylalanine (L-dopa). L-dopa is decarboxylated to form dopamine through the catalysis of dopa decarboxylase. Dopamine β-hydroxylase β-hydroxylates dopamine to yield noradrenaline. Noradrenaline is N-methylated by phenylethanolamine N-methyltransferase to form adrenaline in adrenergic neurons and other adrenaline

HO

*Dopamine*

*Noradrenaline*

HO

HO

O

OH

CH3

CH3

NH2

NH2

OH

*hydroxylase*

*Dopamine*  

NH2

*Dopa decarboxylase*

O

OH

*Tyrosine Tyrosine hydroxylase L-dopa*

NH2

CH3

N

OH

CH3 <sup>H</sup>

*Adrenaline N-methyltransferase*

2006, Baynes and Dominiczak, 2007, Rang et al., 2007).

HO

HO

producing cells.

HO

NA, studies on distinct central nervous system regions revealed that the distribution of dopamine and NA neurons is markedly different (Bloom, 2006). More than half of the central nervous system catecholamine content is dopamine and extremely large amounts are found in the basal ganglia (especially the *caudate nucleus*), the *nucleus accumbens*, the olfactory tubercle, the central nucleus of the amygdala, the median eminence, and restricted fields of the frontal cortex (Bloom, 2006). Of the wide variety of connections, the greatest attention has been given to the long projections between the major dopaminecontaining nuclei located at the substantia nigra and ventral tegmentum and their targets in the striatum. In addition, two important central dopaminergic systems are functionally located outside the blood-brain barrier: in the chemoceptor trigger zone and in the anterior pituitary. In the periphery, dopamine is synthesized in epithelial cells of the proximal tubules and it is thought to exert local diuretic and natriuretic effects in the kidneys (Esler et al., 1990, Bloom, 2006). Likewise, dopamine receptors are found in the proximal gastrointestinal tract where their activation delays gastric emptying. Dopaminergic receptors are also present in renal, mesenteric, coronary and intracerebral arteries (Bloom, 2006). At the cellular level, the actions of dopamine depend on the expression of the different receptor subtypes and the contingent actions of other transmitters to the same target neurons (Bloom, 2006). This catecholamine will not be deeply addressed in the present work, except in specific circumstances where it can be of relevance to the theme.

#### **4. Synthesis of catecholamines**

The mechanisms of catecholamine synthesis, storage, release, and binding have been mainly studied in sympathetically innervated organs and in the adrenal medulla (Westfall and Westfall, 2006).

The steps that occur in the synthesis of dopamine, NA, and ADR are shown in Figure 2. In the cytoplasm, tyrosine is sequentially 3-hydroxylated by tyrosine hydroxylase to form L-3,4-dihydroxyphenylalanine, which is then decarboxylated to form dopamine by L-3,4 dihydroxyphenylalanine decarboxylase (also known as aromatic L-amino acid decarboxylase) (Axelrod, 1962). Dopamine β-hydroxylase β-hydroxylates dopamine to yield NA, which is N-methylated by PNMT to form ADR, in adrenergic neurons and other ADR producing cells (Rang et al., 2007). The enzymes involved in this synthesis have been identified, cloned, and characterized (Nagatsu, 1991).

In adrenergic neurons, the enzymes that participate in NA formation are synthesized in the cell bodies of neurons and are then transported along the axon to the terminals. In the course of NA synthesis, both the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine and the decarboxylation of L-3,4-dihydroxyphenylalanine to dopamine take place in the cytoplasm. About half of the dopamine formed in the cytoplasm is then actively transported into the dopamine β-hydroxylase-containing storage vesicles, where it is converted to NA. The remainder escapes active transport to the vesicles and is deaminated to 3,4 dihydroxyphenylacetic acid and subsequently *O*-methylated to homovanillic acid (HVA) (Westfall and Westfall, 2006). In ADR-producing cells, the NA formed in the vesicles leaves them and it is methylated in the cytoplasm by PNMT to yield ADR. ADR then re-enters the

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 7

Neurotransmission depends mainly on the regulated release of chemical transmitter molecules. That release requires the packing of these substances into the specialized secretory vesicles of neurons and neuroendocrine cells. The neuronal content of catecholamines is confined to vesicles, while in adrenal medulla, catecholamines are stored in chromaffin granules. These vesicles contain extremely high concentrations of catecholamines (approximately 21% dry weight), ascorbic acid, and adenosine-5' triphosphate (ATP), as well as chromogranins, dopamine β-hydroxylase, and peptides, including enkephalin and neuropeptide Y. Two types of storage vesicles are actually found in sympathetic nerve terminals: large dense-core vesicles equivalent to the chromaffin granules and small dense-core vesicles containing NA, ATP, and membrane-bound dopamine β-hydroxylase (Bloom, 2006). The enhanced activity of the sympathetic nervous system is accompanied by increase of both dopamine β-hydroxylase and chromogranins in circulation, thus supporting the argument that the process of release, following the

adrenergic nerve stimulation, involves exocytosis (Westfall and Westfall, 2006).

The storage process decreases the intraneuronal metabolism of the transmitters and their leak out of the cell. This allows to control the concentration gradient across the plasma membrane and to prevent possible toxic effects that could occur when the cytoplasmic concentration of catecholamines exceeds critical levels (Schuldiner, 1994, Masson et al., 1999). The storage process is mediated by specific vesicular transporters (Schuldiner et al., 1978, Erickson et al., 1992, Schuldiner, 1994, Masson et al., 1999). In mammals, there are two closely related vesicular monoamine transporter (VMAT) isoforms that are named VMAT-1 and VMAT-2. VMAT-1 is primarily present in endocrine and paracrine cells, while VMAT-2 is the predominant monoamine transporter in the nervous system (Masson et al., 1999). VMAT-2 is mainly expressed in dense core vesicles of axon terminals (Figure 3) (Nirenberg

Monoamine vesicular transporters are relatively promiscuous when concerning to their substrates. They transport dopamine, NA, ADR, and 5-HT but they differ in substrate preference and affinity. Several vesicular transport complementary deoxyribonucleic acid (cDNA) have been cloned; these complementary DNAs reveal open reading frames predictive of proteins with 12 transmembrane domains (Masson et al., 1999). Reserpine inhibits the monoamine vesicular transport and ultimately leads to the depletion of

The vesicular transport is driven by pH and potential gradients which are established by ATP-dependent proton pumps. The ATP-driven H+ pump, on one hand, acidifies the vesicular lumen (ΔpH) and, on the other hand, generates a potential gradient (ΔΨ)(Schuldiner et al., 1978, Johnson and Scarpa, 1979). For every molecule taken in, two internal H+ ions are extruded by VMAT-2 by an antiport process (Masson et al., 1999). Therefore, the intravesicular concentration of monoamines depends upon ΔΨ and ΔpH (Njus et al., 1986, Rottenberg, 1986, Johnson, 1988, Schuldiner, 1994, Masson et al., 1999). In addition, the monoamine uptake is modulated by vesicle-associated heterotrimeric guanine nucleotide binding regulatory proteins (G proteins), Gαo2 and Gαq (Ahnert-Hilger et al., 1998, Holtje et al., 2000, Holtje et al., 2003). G proteins control transmitter storage: the

catecholamines inside the nerve endings (von Euler, 1972, Masson et al., 1999).

**5. Intracellular storage of catecholamines** 

et al., 1997).

vesicles, where it is stored and concentrated for subsequent release (Masson et al., 1999, Westfall and Westfall, 2006).

The hydroxylation of tyrosine by tyrosine hydroxylase is generally regarded as the ratelimiting step in the biosynthesis of catecholamines (Zigmond et al., 1989). This enzyme is activated after stimulation of sympathetic nerves or adrenal medulla and is tightly regulated. In fact, tyrosine hydroxylase is substrate for protein kinase A (PKA), protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII). The kinasecatalysed phosphorylation results in the increase of tyrosine hydroxylase activity (Zigmond et al., 1989, Daubner et al., 1992). This is an important acute mechanism to increase the synthesis of catecholamines in response to elevated nerve stimulation. Likewise, tyrosine hydroxylase activity is subject to a feedback inhibition by catechol compounds, which allosterically modulate the activity of the enzyme (Kumer and Vrana, 1996). On the other hand, the expression of tyrosine hydroxylase can be increased at multiple levels, including transcription, ribonucleic acid (RNA) processing, regulation of RNA stability, translation, and enzyme stability (Kumer and Vrana, 1996).

Tyrosine hydroxylase deficiency has been reported in humans and is characterized by generalized rigidity, hypokinesia, among other symptoms. Low cerebrospinal fluid levels of NA and dopamine metabolites, like HVA and 3-methoxy-4-hydroxy-phenylethylene glycol are observed in humans with tyrosine hydroxylase deficiency (Wevers et al., 1999, Carson and Robertson, 2002). The tyrosine hydroxylase knockout is unviable in mice as they die in the embryonic stage, presumably because catecholamine loss results in altered cardiac function (Zhou et al., 1995). Interestingly, residual levels of dopamine are present in these embryonic mice, suggesting that tyrosinase may be an alternate way to form catecholamines. However, the amount of tyrosinase-derived catecholamines is clearly not sufficient for the survival of the animals (Carson and Robertson, 2002).

Dopamine β-hydroxylase deficiency in humans is characterized by orthostatic hypotension, ptosis of the eyelids, retrograde ejaculation, and elevated plasma levels of dopamine (Westfall and Westfall, 2006). In the case of dopamine β-hydroxylase-deficient mice, there is about 90% embryonic mortality (Carson and Robertson, 2002).

At rest, the basal cardiovascular function in PNMT knockout mice showed little change. In fact, ADR was found indispensable for normal blood pressure and cardiac filling responses to stress but it was not required in tachycardia during stress or in normal cardiovascular function at rest (Bao et al., 2007, Sun et al., 2008). Furthermore, glucocorticoids levels are very important in the expression of PMNT and thus in the rate of ADR synthesis. Glucocorticoids are very relevant in the size of the stores of ADR available for release in the brain (Moore and Phillipson, 1975b, a), adrenal medulla (Kelner and Pollard, 1985, Stachowiak et al., 1988, Wan and Livett, 1989, Ross et al., 1990, Betito et al., 1992, Wong et al., 1992), heart (Kennedy and Ziegler, 1991, Krizanova et al., 2001, Kvetnansky et al., 2004) or lungs (Kennedy et al., 1993). The activities of both tyrosine hydroxylase and dopamine βhydroxylase are also increased in the adrenal medulla under the influence of glucocorticoids (Carroll et al., 1991). Thus, any stress that persists sufficiently to evoke an enhanced secretion of corticotrophin mobilizes the appropriate hormones of both the adrenal cortex (predominantly cortisol in humans) and adrenal medulla.

### **5. Intracellular storage of catecholamines**

6 Neuroscience – Dealing with Frontiers

vesicles, where it is stored and concentrated for subsequent release (Masson et al., 1999,

The hydroxylation of tyrosine by tyrosine hydroxylase is generally regarded as the ratelimiting step in the biosynthesis of catecholamines (Zigmond et al., 1989). This enzyme is activated after stimulation of sympathetic nerves or adrenal medulla and is tightly regulated. In fact, tyrosine hydroxylase is substrate for protein kinase A (PKA), protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII). The kinasecatalysed phosphorylation results in the increase of tyrosine hydroxylase activity (Zigmond et al., 1989, Daubner et al., 1992). This is an important acute mechanism to increase the synthesis of catecholamines in response to elevated nerve stimulation. Likewise, tyrosine hydroxylase activity is subject to a feedback inhibition by catechol compounds, which allosterically modulate the activity of the enzyme (Kumer and Vrana, 1996). On the other hand, the expression of tyrosine hydroxylase can be increased at multiple levels, including transcription, ribonucleic acid (RNA) processing, regulation of RNA stability, translation,

Tyrosine hydroxylase deficiency has been reported in humans and is characterized by generalized rigidity, hypokinesia, among other symptoms. Low cerebrospinal fluid levels of NA and dopamine metabolites, like HVA and 3-methoxy-4-hydroxy-phenylethylene glycol are observed in humans with tyrosine hydroxylase deficiency (Wevers et al., 1999, Carson and Robertson, 2002). The tyrosine hydroxylase knockout is unviable in mice as they die in the embryonic stage, presumably because catecholamine loss results in altered cardiac function (Zhou et al., 1995). Interestingly, residual levels of dopamine are present in these embryonic mice, suggesting that tyrosinase may be an alternate way to form catecholamines. However, the amount of tyrosinase-derived catecholamines is clearly not

Dopamine β-hydroxylase deficiency in humans is characterized by orthostatic hypotension, ptosis of the eyelids, retrograde ejaculation, and elevated plasma levels of dopamine (Westfall and Westfall, 2006). In the case of dopamine β-hydroxylase-deficient mice, there is

At rest, the basal cardiovascular function in PNMT knockout mice showed little change. In fact, ADR was found indispensable for normal blood pressure and cardiac filling responses to stress but it was not required in tachycardia during stress or in normal cardiovascular function at rest (Bao et al., 2007, Sun et al., 2008). Furthermore, glucocorticoids levels are very important in the expression of PMNT and thus in the rate of ADR synthesis. Glucocorticoids are very relevant in the size of the stores of ADR available for release in the brain (Moore and Phillipson, 1975b, a), adrenal medulla (Kelner and Pollard, 1985, Stachowiak et al., 1988, Wan and Livett, 1989, Ross et al., 1990, Betito et al., 1992, Wong et al., 1992), heart (Kennedy and Ziegler, 1991, Krizanova et al., 2001, Kvetnansky et al., 2004) or lungs (Kennedy et al., 1993). The activities of both tyrosine hydroxylase and dopamine βhydroxylase are also increased in the adrenal medulla under the influence of glucocorticoids (Carroll et al., 1991). Thus, any stress that persists sufficiently to evoke an enhanced secretion of corticotrophin mobilizes the appropriate hormones of both the adrenal cortex

sufficient for the survival of the animals (Carson and Robertson, 2002).

about 90% embryonic mortality (Carson and Robertson, 2002).

(predominantly cortisol in humans) and adrenal medulla.

Westfall and Westfall, 2006).

and enzyme stability (Kumer and Vrana, 1996).

Neurotransmission depends mainly on the regulated release of chemical transmitter molecules. That release requires the packing of these substances into the specialized secretory vesicles of neurons and neuroendocrine cells. The neuronal content of catecholamines is confined to vesicles, while in adrenal medulla, catecholamines are stored in chromaffin granules. These vesicles contain extremely high concentrations of catecholamines (approximately 21% dry weight), ascorbic acid, and adenosine-5' triphosphate (ATP), as well as chromogranins, dopamine β-hydroxylase, and peptides, including enkephalin and neuropeptide Y. Two types of storage vesicles are actually found in sympathetic nerve terminals: large dense-core vesicles equivalent to the chromaffin granules and small dense-core vesicles containing NA, ATP, and membrane-bound dopamine β-hydroxylase (Bloom, 2006). The enhanced activity of the sympathetic nervous system is accompanied by increase of both dopamine β-hydroxylase and chromogranins in circulation, thus supporting the argument that the process of release, following the adrenergic nerve stimulation, involves exocytosis (Westfall and Westfall, 2006).

The storage process decreases the intraneuronal metabolism of the transmitters and their leak out of the cell. This allows to control the concentration gradient across the plasma membrane and to prevent possible toxic effects that could occur when the cytoplasmic concentration of catecholamines exceeds critical levels (Schuldiner, 1994, Masson et al., 1999). The storage process is mediated by specific vesicular transporters (Schuldiner et al., 1978, Erickson et al., 1992, Schuldiner, 1994, Masson et al., 1999). In mammals, there are two closely related vesicular monoamine transporter (VMAT) isoforms that are named VMAT-1 and VMAT-2. VMAT-1 is primarily present in endocrine and paracrine cells, while VMAT-2 is the predominant monoamine transporter in the nervous system (Masson et al., 1999). VMAT-2 is mainly expressed in dense core vesicles of axon terminals (Figure 3) (Nirenberg et al., 1997).

Monoamine vesicular transporters are relatively promiscuous when concerning to their substrates. They transport dopamine, NA, ADR, and 5-HT but they differ in substrate preference and affinity. Several vesicular transport complementary deoxyribonucleic acid (cDNA) have been cloned; these complementary DNAs reveal open reading frames predictive of proteins with 12 transmembrane domains (Masson et al., 1999). Reserpine inhibits the monoamine vesicular transport and ultimately leads to the depletion of catecholamines inside the nerve endings (von Euler, 1972, Masson et al., 1999).

The vesicular transport is driven by pH and potential gradients which are established by ATP-dependent proton pumps. The ATP-driven H+ pump, on one hand, acidifies the vesicular lumen (ΔpH) and, on the other hand, generates a potential gradient (ΔΨ)(Schuldiner et al., 1978, Johnson and Scarpa, 1979). For every molecule taken in, two internal H+ ions are extruded by VMAT-2 by an antiport process (Masson et al., 1999). Therefore, the intravesicular concentration of monoamines depends upon ΔΨ and ΔpH (Njus et al., 1986, Rottenberg, 1986, Johnson, 1988, Schuldiner, 1994, Masson et al., 1999). In addition, the monoamine uptake is modulated by vesicle-associated heterotrimeric guanine nucleotide binding regulatory proteins (G proteins), Gαo2 and Gαq (Ahnert-Hilger et al., 1998, Holtje et al., 2000, Holtje et al., 2003). G proteins control transmitter storage: the

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 9

monoamines stored inside the vesicles represent the upstream signal that mediates the inhibition to further uptake through G proteins. In fact, biogenic amines, with the exception of ADR, lead to G protein-mediated inhibition; VMATs have a receptor-like area in their first luminal loop that senses the intravesicular concentration of monoamines (Brunk et al.,

Depolarization of the axonal terminal triggers the release of catecholamines into the cleft. The content of the vesicles, including enzymes, neurotransmitters, and hormones, is discharged to the exterior through a process termed exocytosis (Figure 3)*.* Triggered exocytosis is the most common cellular mechanism for the release of polar molecules and it involves several mechanisms that include vesicle docking, priming, and fusion. It can be regulated or constitutive. In synaptic transmission, a synchronized mechanism provides the cells with a way for precisely timed release of molecules into the extracellular space (Pocock and Richards, 2006). The full sequence of steps by which the nerve impulse affects the

In the adrenal medulla, the triggering event to exocytosis is the release of acetylcholine by the preganglionic fibres; that acetylcholine binds to nicotinic receptors on chromaffin cells which produce a localized depolarization. In most types of cells, regulated exocytosis is triggered by the increase in cytosolic free calcium (Ca2+) (Meir et al., 1999, Pocock and Richards, 2006). This cytosolic Ca2+ enters the cells from the extracellular medium, through the voltage-gated Ca2+ channels. The voltage-gated Ca2+ channels open following depolarization of the plasma membrane, making the Ca2+ able to diffuse into the cell down its electrochemical gradient. Ca2+ is also mobilized from intracellular stores (mainly the endoplasmic reticulum). As result, the intracellular free Ca2+ increases and it triggers the fusion of secretory vesicles with the plasma membrane. Ca2+-triggered secretion involves the interaction of highly conserved molecular scaffolding proteins and leads to the docking of the vesicles (Aunis, 1998, Meir et al., 1999). As fusion continues, a pore is formed and it connects the extracellular space with the interior of the vesicle thus providing a pathway for vesicle content to diffuse into the extracellular space (Pocock and Richards, 2006) (Figure 3). Other aspects concerning modulation of catecholamine transmission or secretion will be better addressed in a subsequent section where adrenoceptors and their role in augmenting

release of transmitters from sympathetic neurons is not yet fully understood.

or diminishing the synaptic release will be focused.

**7. Neuronal transporters involved in catecholamine reuptake** 

Synaptic transmission involves the regulated release of transmitters into the synaptic cleft, where they interact with receptors that subsequently transduce the information. The removal of catecholamines from the cleft usually occurs by uptake either back to the presynaptic terminal or into the postsynaptic cell (Iversen et al., 1967, Axelrod and Kopin, 1969). A number of high-affinity transporters have been identified, namely for dopamine, NA, 5-HT, and several aminoacid transmitters (Masson et al., 1999). These transporters are members of an extensive family that share common structural motifs, particularly the putative 12-transmembrane helices (Bönisch and Brüss, 2006). The transporters show a similar configuration of intracellular and extracellular loops or related regions that contain

2006).

**6. Release of catecholamines** 

Fig. 3. Catecholamines in axon terminals. The axon terminal has neurotransmitter storage vesicles with catecholamines and cotransmitters. The vesicular monoamine transporter (VMAT) is responsible for the transport of catecholamines to the storage vesicles, maintaining their cytosolic concentration low. After catecholamine release to the synaptic cleft, catecholamines are reuptaken to the presynaptic terminal by noradrenaline transporter (NET), and/or taken to extraneuronal cells by the extraneuronal transporters. The most important extraneuronal transporter for catecholamines is extraneuronal monoamine transporter (EMT). Catecholamines are metabolized by intracellular enzymes. Monoamine oxidase (MAO) is located in the outer membrane of mitochondria in neurons and in extraneuronal cells. Catechol-*O*-methyl transferase (COMT) is located at extraneuronal cells. Both enzymes (COMT and MAO) are the main responsible for the metabolism of catecholamines. The enzymatic process leads to the formation of several metabolites. To exert their actions, the catecholamines and other neurotransmitters in the synaptic cleft bind to different pre- and postsynaptic receptors. Such binding leads to alterations in the postsynaptic cell and activation of intracellular pathways through G proteins. In presynaptic neurons, catecholamines bind to autoreceptors and activate feed-back responses that change their own release*.* 

monoamines stored inside the vesicles represent the upstream signal that mediates the inhibition to further uptake through G proteins. In fact, biogenic amines, with the exception of ADR, lead to G protein-mediated inhibition; VMATs have a receptor-like area in their first luminal loop that senses the intravesicular concentration of monoamines (Brunk et al., 2006).
