**9. Metabolism and action termination**

12 Neuroscience – Dealing with Frontiers

All three OCTs can transport catecholamines in addition to a wide variety of other organic acids, including 5-HT, histamine, choline, spermine, guanidine, and creatinine (Eisenhofer, 2001). The affinity of extraneuronal monoamine transporter for NA is rather low (Km > 0.5 mM), which is compensated by its high capacity (high turnover number). The transport efficiency of OCT1 and OCT2 for dopamine, NA, ADR, and 5-HT is usually

OCTs share common features. A single positive charge is required to be an OCT substrate, while uncharged, doubly charged or negatively charged substances are not transported by OCTs (Schömig et al., 2006). A decrease in extracellular pH and the depolarization of the cytoplasmatic membrane will reduce the OCT activity. The OCT mediated transport is independent of Na+ and Cl<sup>−</sup> gradients and all three OCTs are transporters, not just channels, as shown by trans-stimulation experiments. OCT1, OCT2 or extraneuronal monoamine transporter may mediate electrogenic uniport of a substrate or electroneutral exchange of two substrates (antiport). The OCTs are relatively resistant to the inhibitors of the neuronal transporters such as desipramine (Eisenhofer et al., 1991, Schömig et al., 2006), but are inhibited by steroids, such as corticosterone, and the *O*-methylated metabolites of catecholamines, like normetanephrine and metanephrine (Eisenhofer, 2001, Costa et al., 2009a, Nissinen and Männistö, 2010). Other compounds, like GF120918, were shown to inhibit extraneuronal monoamine transporter at low concentrations in isolated rat

Extraneuronal monoamine transporter (OCT3) is expressed in many but not all tissues. The expression varies greatly between organs and during organ development (e.g. in placenta). Consistently, high expression has been reported for placenta and heart (Eisenhofer, 2001). Other reports indicate high extraneuronal monoamine transporter expression in the area postrema (Haag et al., 2004, Vialou et al., 2004) and very low in the kidneys (Eisenhofer, 2001). Interestingly, extraneuronal monoamine transporter expression has been also

For NA, the uptake by NET is more relevant for signal termination than extraneuronal uptake. It has been estimated that the sympathetic nerves remove approximately 87% of released NA by NET, while 5% is removed by extraneuronal monoamine transporter. The remainder 8% is diffused into the circulation (Eisenhofer, 2001). This data, however, does not necessarily reflect the relative importance of the two processes. The proximity of the neuronal transporters to the location of catecholamine release (when compared to extraneuronal transporters) implies that the transmitter removed by extraneuronal transport has a higher duration and wider range of action. Extraneuronal uptake, therefore, may be particularly important for the removal of neuronal released catecholamines in tissues where high concentrations of adrenergic receptors exist (Eisenhofer, 2001). The clearance of circulating catecholamines is primarily mediated by non neuronal mechanisms, with liver and kidney accounting for over 60% (Eisenhofer, 2001, Westfall and Westfall, 2006). As stated above, most NA released by sympathetic nerves is removed by neuronal reuptake, which is in contrast with that of ADR. ADR when secreted directly into the bloodstream from the adrenal medulla is predominantly inactivated by extraneuronal uptake and metabolism. ADR has lower affinity for neuronal uptake than NA and consequently the neuronal process contributes approximately 50% less for its elimination when compared with NA (Iversen, 1965a). In contrast, ADR is removed by extraneuronal uptake 2-3 times

suggested to occur in neurons (Kristufek et al., 2002, Shang et al., 2003).

more efficiently than NA (Iversen, 1965b, Eisenhofer et al., 1992b).

low (Schömig et al., 2006).

cardiomyocytes (Costa et al., 2009a).

The pharmacological actions of NA and ADR are terminated by: (i) reuptake into nerve terminals by NET; (ii) uptake at extraneuronal sites by extraneuronal monoamine transporter, OCT1, or OCT2; and (iii) metabolic transformation. Catecholamines undergo a complex metabolic fate, mediated by several enzymes, including aldehyde reductase, aldose reductase, aldehyde dehydrogenase, alcohol dehydrogenase, catechol-*O*-methyltransferase (COMT), dopamine β-hydroxylase, monoamine oxidase (MAO) type A and B, monoaminepreferring phenolsulfotransferase, and PNMT (Dooley, 1998, Goldstein et al., 2003). *In vivo,* two of the most important enzymes responsible for the metabolic transformation of catecholamines are MAO and COMT (Westfall and Westfall, 2006).

#### **9.1 Monoaminoxidase metabolism**

MAO was first described as tyramine oxidase by Mary Hare-Bernheim in 1928, since it catalyses the oxidative deamination of tyramine. This enzyme was found to oxidize several monoamines, including catecholamines, i.e. dopamine, NA, and ADR, and also 5-HT. MAO [amine: oxygen oxidoreductase (deaminating)] catalyses the following reaction:

$$\text{RCH}\_2\text{NH}\_2 + \text{H}\_2\text{O} + \text{O}\_2 \rightarrow \text{RCHO} + \text{NH}\_3 + \text{H}\_2\text{O}\_2$$

MAO acts on primary amines and also on some secondary and tertiary amines (Nagatsu, 1991). It is located in the outer membrane of mitochondria (Schnaitman et al., 1967) both in neuronal and extraneuronal cells (Trendelenburg, 1988) (Figure 3). It is a flavo-protein, with flavin adenine dinucleotide as cofactor (Kearney et al., 1971). Two forms of MAO exist, i.e. MAO-A and MAO-B (Johnston, 1968). In humans, MAO-A is abundant in the brain, liver, and in the syncytiotrophoblast layer of term placenta, whereas liver, lungs, platelets, lymphocytes, osteocytes that surround the blood vessels, and the intestine are rich in MAO-B (Abell and Kwan, 2001, Nagatsu, 2004). In the human brain, MAO-A is located in the catecholamine-containing regions, with the highest levels being found in *locus ceruleus*. MAO-B is prominent in the dorsal *raphe nuclei*, which are known to have serotonergic neurons. MAO-B is also present in the posterior hypothalamus and in glial cells (Abell and Kwan, 2001, Nagatsu, 2004).

5-HT, NA, and ADR are preferential substrates for MAO-A, and clorgyline and Ro 41-1049 are MAO-A inhibitors (Johnston, 1968, Kitaichi et al., 2010); MAO-B prefers βphenylethylamine as a substrate and it is inhibited by deprenyl and lazabemide (Knoll et al., 1978, Kitaichi et al., 2010). Dopamine, tyramine, and tryptamine are oxidized with equal affinity by MAO-A and MAO-B (Glover et al., 1977). Pargyline inhibits both MAO-A and

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 15

COMT include L-3,4-dihydroxyphenylalanine, all three endogenous catecholamines (dopamine, NA, and ADR), their hydroxylated metabolites, catecholestrogens, ascorbic acid, and dihydroxyindolic intermediates of melanin (Mannisto and Kaakkola, 1999, Nissinen

In COMT knockout mice, minor behaviour changes were detected and the neurochemistry of catecholamines in the brain is virtually unaltered (Gogos et al., 1998). Mutant mice showed sexually dimorphic and region-specific changes of dopamine levels, notably in the frontal cortex. Mutant male mice have almost a three-fold increase of dopamine levels in the frontal cortex, while no changes were observed in the striatum or hypothalamus. In mutant female mice no alterations in dopamine levels were observed when compared to the wild type (Gogos et al., 1998). Despite the complete lack of *comt* gene in homozygous mice, residual HVA levels were detectable in several brain areas, revealing a possible and still unidentified methylation pathway in the brain (Gogos et al., 1998). Female knockout mice showed impaired emotional reactivity, while heterozygous males were more aggressive (Gogos et al., 1998). The work by Gogos and colleagues suggested that the importance of COMT in the emotional and social behaviour has probably been undervalued and the complete lack of COMT can be partially compensated, at least in

**9.2.1 The cross-talk between catecholamine catabolic enzymes and transporters** 

combined lack of MAO-A and MAO-B (Lenders et al., 1996).

Of relevance, the main mechanism that reduces the life span of catecholamines in the extracellular space is their uptake by active transport and not their enzymatic metabolism. It has been shown that NET inhibitors (e.g., cocaine, imipramine, and desipramine) potentiate the effects of the neurotransmitters, while inhibitors of MAO and COMT have relatively little immediate effect (Gonçalves et al., 1989, Eisenhofer, 1994, Eisenhofer and Finberg, 1994, Friedgen et al., 1994, Blaha et al., 1996, Friedgen et al., 1996). Early studies in isolated tissues showed that the inhibition of MAO or COMT could limit the ability of neuronal and extraneuronal uptake to clear extracellular catecholamines (Furchgott and Garcia, 1968, Belfrage et al., 1977). More recent *in vivo* studies however indicate that inhibition of one enzyme under normal physiological conditions has little effect on the overall catecholamine clearance and on their extracellular or circulating levels (Eisenhofer, 1994, Eisenhofer and Finberg, 1994, Friedgen et al., 1994, Blaha et al., 1996, Friedgen et al., 1996). Only when MAO and COMT are both inhibited or subjected to saturating concentrations of substrate, the clearance of catecholamines is significantly impaired (Eisenhofer, 1994, Friedgen et al., 1996). To confirm the redundancy of metabolizing systems, in MAO-A-deficient humans or rats a marked decrease in deaminated catecholamine metabolites and a concomitant marked elevation of *O*-methylated amine metabolites occur (Eisenhofer and Finberg, 1994, Lenders et al., 1996). These neurochemical changes are only slightly exaggerated in patients with

The activity of metabolizing enzymes can influence the catecholamine net transport and the extracellular levels of catecholamines. Moreover, the metabolizing enzymes are always required for the irreversible chemical alteration of catecholamines following neuronal or extraneuronal uptake. Nevertheless, Trendelenburg stated that understanding the

and Männistö, 2010).

mice (Gogos et al., 1998).

MAO-B (Blaha et al., 1996, Carvalho et al., 2001, Carmo et al., 2003, Duarte et al., 2004). MAO-A appears to be the main enzyme in the metabolism of 5-HT and NA, while the location of MAO-B, abundant in serotonergic neurons, remains under debate (Abell and Kwan, 2001).

In the brain of MAO-A-deficient mouse pups, NA levels increased up to two-fold, while a small increase in dopamine levels was observed. Borderline mental retardation and abnormal behavioural are observed in humans with selective MAO-A deficiency. Severe mental retardation in patients with combined MAO-A/MAO-B deficiency and Norrie disease are also observed (Lenders et al., 1996). Human males from a Dutch family with a complete deletion of the *mao-a* gene in the X chromosome were reported to show abnormal aggressive behaviour (Brunner et al., 1993), as observed in adult mice (Cases et al., 1995). MAO-A-deficient adult male mice show enhanced aggressive behaviour and enhanced emotional learning, probably due to elevated levels of 5-HT in the brain in the pup stage (Cases et al., 1995, Kim et al., 1997). Furthermore, MAO-A knockout mice exhibited a dramatic reduction of defensive and fear-related behaviours in the presence of predatorrelated cues, such as predator urine or an anaesthetized rat (Godar et al., 2010).

All regions of the brain of MAO-B-deficient mouse pups showed increased levels of βphenylethylamine (Lenders et al., 1996), especially in striatum and prefrontal cortex (Bortolato et al., 2009). The MAO-B knockout mice show behavioural disinhibition and decrease anxiety-like responses partially through a regional increase in β-phenylethylamine levels (Bortolato et al., 2009). In contrast to the borderline mental retardation and abnormal behavioural phenotype in subjects with selective MAO-A deficiency and the severe mental retardation in patients with combined MAO-A/MAO-B deficiency and Norrie disease, the MAO-B-deficient human subjects neither exhibited abnormal behaviour nor mental retardation except increased reactivity to stress (Grimsby et al., 1997). The subjects with the *mao-b* gene deletion have complete absence of platelet MAO-B activity and have increased brain levels and urinary excretion of β-phenylethylamine (Lenders et al., 1996). In fact, the inhibition of MAO-B indicates that this form of MAO has a lower contribution to the metabolism of endogenous catecholamines and of their *O*-methylated metabolites (Eisenhofer and Finberg, 1994).

#### **9.2 Catechol-***O***-methyl transferase metabolism**

Another important catecholamine-metabolizing enzyme (COMT) was first described and purified by Axelrod (Axelrod, 1958, Axelrod and Tomchick, 1958) (Figure 3). One single gene for COMT codes for both soluble COMT and membrane-bound COMT. That gene has different transcription starting sites. COMT is an intracellular enzyme, the most abundant form being the soluble COMT with a minor fraction as membrane bound COMT (Mannisto and Kaakkola, 1999). COMT catalyses the transfer of the methyl group of *S*-adenosyl-Lmethionine to one of the hydroxyl groups in the catechol in the presence of magnesium ion (Mg2+) (Mannisto and Kaakkola, 1999). COMT is widely distributed throughout the body. High levels of COMT are found in the liver, proximal tubular epithelial cells of the kidneys, and other extraneuronal cells, namely adrenomedullary chromaffin cells (Eisenhofer et al., 1998, Mannisto and Kaakkola, 1999). However, little or no COMT is found in sympathetic neurons. In the presynaptic terminals of the brain, no significant COMT is detected, but it is reported in some postsynaptic neurons and glial cells. The physiological substrates of

MAO-B (Blaha et al., 1996, Carvalho et al., 2001, Carmo et al., 2003, Duarte et al., 2004). MAO-A appears to be the main enzyme in the metabolism of 5-HT and NA, while the location of MAO-B, abundant in serotonergic neurons, remains under debate (Abell and

In the brain of MAO-A-deficient mouse pups, NA levels increased up to two-fold, while a small increase in dopamine levels was observed. Borderline mental retardation and abnormal behavioural are observed in humans with selective MAO-A deficiency. Severe mental retardation in patients with combined MAO-A/MAO-B deficiency and Norrie disease are also observed (Lenders et al., 1996). Human males from a Dutch family with a complete deletion of the *mao-a* gene in the X chromosome were reported to show abnormal aggressive behaviour (Brunner et al., 1993), as observed in adult mice (Cases et al., 1995). MAO-A-deficient adult male mice show enhanced aggressive behaviour and enhanced emotional learning, probably due to elevated levels of 5-HT in the brain in the pup stage (Cases et al., 1995, Kim et al., 1997). Furthermore, MAO-A knockout mice exhibited a dramatic reduction of defensive and fear-related behaviours in the presence of predator-

related cues, such as predator urine or an anaesthetized rat (Godar et al., 2010).

All regions of the brain of MAO-B-deficient mouse pups showed increased levels of βphenylethylamine (Lenders et al., 1996), especially in striatum and prefrontal cortex (Bortolato et al., 2009). The MAO-B knockout mice show behavioural disinhibition and decrease anxiety-like responses partially through a regional increase in β-phenylethylamine levels (Bortolato et al., 2009). In contrast to the borderline mental retardation and abnormal behavioural phenotype in subjects with selective MAO-A deficiency and the severe mental retardation in patients with combined MAO-A/MAO-B deficiency and Norrie disease, the MAO-B-deficient human subjects neither exhibited abnormal behaviour nor mental retardation except increased reactivity to stress (Grimsby et al., 1997). The subjects with the *mao-b* gene deletion have complete absence of platelet MAO-B activity and have increased brain levels and urinary excretion of β-phenylethylamine (Lenders et al., 1996). In fact, the inhibition of MAO-B indicates that this form of MAO has a lower contribution to the metabolism of endogenous catecholamines and of their *O*-methylated metabolites

Another important catecholamine-metabolizing enzyme (COMT) was first described and purified by Axelrod (Axelrod, 1958, Axelrod and Tomchick, 1958) (Figure 3). One single gene for COMT codes for both soluble COMT and membrane-bound COMT. That gene has different transcription starting sites. COMT is an intracellular enzyme, the most abundant form being the soluble COMT with a minor fraction as membrane bound COMT (Mannisto and Kaakkola, 1999). COMT catalyses the transfer of the methyl group of *S*-adenosyl-Lmethionine to one of the hydroxyl groups in the catechol in the presence of magnesium ion (Mg2+) (Mannisto and Kaakkola, 1999). COMT is widely distributed throughout the body. High levels of COMT are found in the liver, proximal tubular epithelial cells of the kidneys, and other extraneuronal cells, namely adrenomedullary chromaffin cells (Eisenhofer et al., 1998, Mannisto and Kaakkola, 1999). However, little or no COMT is found in sympathetic neurons. In the presynaptic terminals of the brain, no significant COMT is detected, but it is reported in some postsynaptic neurons and glial cells. The physiological substrates of

Kwan, 2001).

(Eisenhofer and Finberg, 1994).

**9.2 Catechol-***O***-methyl transferase metabolism** 

COMT include L-3,4-dihydroxyphenylalanine, all three endogenous catecholamines (dopamine, NA, and ADR), their hydroxylated metabolites, catecholestrogens, ascorbic acid, and dihydroxyindolic intermediates of melanin (Mannisto and Kaakkola, 1999, Nissinen and Männistö, 2010).

In COMT knockout mice, minor behaviour changes were detected and the neurochemistry of catecholamines in the brain is virtually unaltered (Gogos et al., 1998). Mutant mice showed sexually dimorphic and region-specific changes of dopamine levels, notably in the frontal cortex. Mutant male mice have almost a three-fold increase of dopamine levels in the frontal cortex, while no changes were observed in the striatum or hypothalamus. In mutant female mice no alterations in dopamine levels were observed when compared to the wild type (Gogos et al., 1998). Despite the complete lack of *comt* gene in homozygous mice, residual HVA levels were detectable in several brain areas, revealing a possible and still unidentified methylation pathway in the brain (Gogos et al., 1998). Female knockout mice showed impaired emotional reactivity, while heterozygous males were more aggressive (Gogos et al., 1998). The work by Gogos and colleagues suggested that the importance of COMT in the emotional and social behaviour has probably been undervalued and the complete lack of COMT can be partially compensated, at least in mice (Gogos et al., 1998).

#### **9.2.1 The cross-talk between catecholamine catabolic enzymes and transporters**

Of relevance, the main mechanism that reduces the life span of catecholamines in the extracellular space is their uptake by active transport and not their enzymatic metabolism. It has been shown that NET inhibitors (e.g., cocaine, imipramine, and desipramine) potentiate the effects of the neurotransmitters, while inhibitors of MAO and COMT have relatively little immediate effect (Gonçalves et al., 1989, Eisenhofer, 1994, Eisenhofer and Finberg, 1994, Friedgen et al., 1994, Blaha et al., 1996, Friedgen et al., 1996). Early studies in isolated tissues showed that the inhibition of MAO or COMT could limit the ability of neuronal and extraneuronal uptake to clear extracellular catecholamines (Furchgott and Garcia, 1968, Belfrage et al., 1977). More recent *in vivo* studies however indicate that inhibition of one enzyme under normal physiological conditions has little effect on the overall catecholamine clearance and on their extracellular or circulating levels (Eisenhofer, 1994, Eisenhofer and Finberg, 1994, Friedgen et al., 1994, Blaha et al., 1996, Friedgen et al., 1996). Only when MAO and COMT are both inhibited or subjected to saturating concentrations of substrate, the clearance of catecholamines is significantly impaired (Eisenhofer, 1994, Friedgen et al., 1996). To confirm the redundancy of metabolizing systems, in MAO-A-deficient humans or rats a marked decrease in deaminated catecholamine metabolites and a concomitant marked elevation of *O*-methylated amine metabolites occur (Eisenhofer and Finberg, 1994, Lenders et al., 1996). These neurochemical changes are only slightly exaggerated in patients with combined lack of MAO-A and MAO-B (Lenders et al., 1996).

The activity of metabolizing enzymes can influence the catecholamine net transport and the extracellular levels of catecholamines. Moreover, the metabolizing enzymes are always required for the irreversible chemical alteration of catecholamines following neuronal or extraneuronal uptake. Nevertheless, Trendelenburg stated that understanding the

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 17

HO

HO

HO

HO

H3CO

H3CO

**Adrenaline**

**COMT**

**MN**

**MAO**

**MOPGAL**

N

OH

N H CH3

OH

O

H CH3

OH

HO

HO

HO

HO

HO

HO

**Noradrenaline**

**MAO**

**DOPEGAL**

**DHPG**

**AR**

N

H H

OH

OH

OH

OH

**COMT**

HO

HO

H3CO

**MHPG**

**ADH and ALDH**

**VMA**

Fig. 4. Representative pathway of the most common metabolic pathways of adrenaline (ADR) and noradrenaline (NA). ADR is synthesized from NA through phenylethanolamine N-methyltransferase (PNMT) catalysis. NA is metabolized preferably by monoamine oxidase (MAO) to dihydroxyphenylglycoaldehyde (DOPEGAL) that is reduced by aldehyde reductase (AR) to form dihydroxyphenylglycol (DHPG). ADR is preferably metabolized by catechol-*O*-methyltransferase (COMT) resulting in metanephrine (MN). MN can be further metabolized by MAO to 3-methoxy-4-hydroxyphenyl-glycoaldehyde (MOPGAL) and by aldehyde reductase (AR) to form 3-methoxy-4-hydroxy-phenylethylene glycol (MHPG). MHPG is further metabolized and can be transformed to vanillylmandelic acid (VMA) by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). VMA is the main catecholamine metabolite in urine (adapted from reference Goldstein et al., 2003).

H3CO

OH

OH

**AR**

COOH

OH

O

interactions and the functions of the active transport and metabolizing processes is very relevant as they function as "pump and leak systems with enzyme(s) inside" (Trendelenburg, 1988, 1990).

An integrated vision explains the results found *in vivo*. In fact, the presence of only one catecholamine-metabolizing enzyme, MAO, in neuronal systems would lead to the assumption that inhibition of this enzyme would impair the catecholamine's neuronal uptake by changing the concentration gradient. However, the additional presence of VMAT-2 maintains the axoplasmic catecholamine levels low. Furthermore, VMAT-2 has higher affinity for NA than MAO, which implies that over 70% of the recaptured NA through NET is sequestered into storage vesicles before being metabolized (Bloom, 2006). Therefore, under normal physiological conditions, the inhibition of MAO has little effect (Graefe and Trendelenburg, 1970, Graefe and Henseling, 1983). Rather than impairing the catecholamine uptake, short-term inhibition of intraneuronal MAO leads to higher retention of recaptured transmitter by VMAT-2 and, thus, an apparent increase in the net uptake of the transmitter (Furchgott and Garcia, 1968). In the long term, if vesicular storage capacity is overwhelmed, inhibition or saturation of intraneuronal MAO can lead to a decrease in neurotransmitter's uptake (Trendelenburg et al., 1972).

#### **9.3 The metabolites of catecholamines**

Complex and dynamic processes are involved in the metabolism of catecholamines, either intracellularly or even after their release into the extracellular fluid. In both neuronal and extraneuronal metabolizing systems, the inactivation of catecholamines occurs in a coordinated fashion, with uptake being followed by metabolism (Graefe and Henseling, 1983). Most of the metabolism of catecholamines takes place in the same cells where they are produced, even before their exocytotic release (Figure 4). Dihydroxyphenylglycol (DHPG, 3,4-dihydroxyphenylethylene glycol) constitutes the main NA metabolite before NA release or reuptake. DHPG is also a deaminated metabolite of ADR (Eisenhofer and Finberg, 1994, Goldstein et al., 2003). DHPG is formed from NA in the cytoplasm of sympathetic nerves by sequential deamination of NA by MAO to form dihydroxyphenylglycoaldehyde. This aldehyde is reduced by aldehyde reductase or aldose reductase to form DHPG or it is oxidized by aldehyde dehydrogenase to form 3,4-dihydroxymandelic acid. DHPG diffuses rapidly across the membrane of the cell into the extracellular fluid and into extraneuronal cells. In the extraneuronal cells, DHPG is metabolized by COMT to form 3-methoxy-4 hydroxy-phenylethylene glycol, or it overflows into the bloodstream (Eisenhofer and Finberg, 1994, Goldstein et al., 2003). Also, the decrease in plasma concentrations of endogenous DHPG and 3,4-dihydroxyphenylacetic acid after inhibition of MAO-A, but not MAO-B, corroborates that NA is a better substrate for the A isoform (Eisenhofer and Finberg, 1994).

COMT has a substantial importance in the metabolism of the deaminated metabolites of NA and dopamine, as corroborated by the increase of plasma levels of 3,4-dihydroxyphenylacetic acid and DHPG after COMT inhibition (Eisenhofer and Finberg, 1994). Alternatively, COMT can catalyse the *O*-methylation of NA to normetanephrine, of ADR to metanephrine, of L-3,4 dihydroxyphenylalanine to 3-methoxytyrosine, and of dopamine mainly to 3 methoxytyramine (Eisenhofer and Finberg, 1994, Goldstein et al., 2003).

interactions and the functions of the active transport and metabolizing processes is very relevant as they function as "pump and leak systems with enzyme(s) inside"

An integrated vision explains the results found *in vivo*. In fact, the presence of only one catecholamine-metabolizing enzyme, MAO, in neuronal systems would lead to the assumption that inhibition of this enzyme would impair the catecholamine's neuronal uptake by changing the concentration gradient. However, the additional presence of VMAT-2 maintains the axoplasmic catecholamine levels low. Furthermore, VMAT-2 has higher affinity for NA than MAO, which implies that over 70% of the recaptured NA through NET is sequestered into storage vesicles before being metabolized (Bloom, 2006). Therefore, under normal physiological conditions, the inhibition of MAO has little effect (Graefe and Trendelenburg, 1970, Graefe and Henseling, 1983). Rather than impairing the catecholamine uptake, short-term inhibition of intraneuronal MAO leads to higher retention of recaptured transmitter by VMAT-2 and, thus, an apparent increase in the net uptake of the transmitter (Furchgott and Garcia, 1968). In the long term, if vesicular storage capacity is overwhelmed, inhibition or saturation of intraneuronal MAO can lead to a decrease in neurotransmitter's

Complex and dynamic processes are involved in the metabolism of catecholamines, either intracellularly or even after their release into the extracellular fluid. In both neuronal and extraneuronal metabolizing systems, the inactivation of catecholamines occurs in a coordinated fashion, with uptake being followed by metabolism (Graefe and Henseling, 1983). Most of the metabolism of catecholamines takes place in the same cells where they are produced, even before their exocytotic release (Figure 4). Dihydroxyphenylglycol (DHPG, 3,4-dihydroxyphenylethylene glycol) constitutes the main NA metabolite before NA release or reuptake. DHPG is also a deaminated metabolite of ADR (Eisenhofer and Finberg, 1994, Goldstein et al., 2003). DHPG is formed from NA in the cytoplasm of sympathetic nerves by sequential deamination of NA by MAO to form dihydroxyphenylglycoaldehyde. This aldehyde is reduced by aldehyde reductase or aldose reductase to form DHPG or it is oxidized by aldehyde dehydrogenase to form 3,4-dihydroxymandelic acid. DHPG diffuses rapidly across the membrane of the cell into the extracellular fluid and into extraneuronal cells. In the extraneuronal cells, DHPG is metabolized by COMT to form 3-methoxy-4 hydroxy-phenylethylene glycol, or it overflows into the bloodstream (Eisenhofer and Finberg, 1994, Goldstein et al., 2003). Also, the decrease in plasma concentrations of endogenous DHPG and 3,4-dihydroxyphenylacetic acid after inhibition of MAO-A, but not MAO-B, corroborates that NA is a better substrate for the A isoform (Eisenhofer and

COMT has a substantial importance in the metabolism of the deaminated metabolites of NA and dopamine, as corroborated by the increase of plasma levels of 3,4-dihydroxyphenylacetic acid and DHPG after COMT inhibition (Eisenhofer and Finberg, 1994). Alternatively, COMT can catalyse the *O*-methylation of NA to normetanephrine, of ADR to metanephrine, of L-3,4 dihydroxyphenylalanine to 3-methoxytyrosine, and of dopamine mainly to 3-

methoxytyramine (Eisenhofer and Finberg, 1994, Goldstein et al., 2003).

(Trendelenburg, 1988, 1990).

uptake (Trendelenburg et al., 1972).

Finberg, 1994).

**9.3 The metabolites of catecholamines** 

Fig. 4. Representative pathway of the most common metabolic pathways of adrenaline (ADR) and noradrenaline (NA). ADR is synthesized from NA through phenylethanolamine N-methyltransferase (PNMT) catalysis. NA is metabolized preferably by monoamine oxidase (MAO) to dihydroxyphenylglycoaldehyde (DOPEGAL) that is reduced by aldehyde reductase (AR) to form dihydroxyphenylglycol (DHPG). ADR is preferably metabolized by catechol-*O*-methyltransferase (COMT) resulting in metanephrine (MN). MN can be further metabolized by MAO to 3-methoxy-4-hydroxyphenyl-glycoaldehyde (MOPGAL) and by aldehyde reductase (AR) to form 3-methoxy-4-hydroxy-phenylethylene glycol (MHPG). MHPG is further metabolized and can be transformed to vanillylmandelic acid (VMA) by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). VMA is the main catecholamine metabolite in urine (adapted from reference Goldstein et al., 2003).

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 19

methoxy-4-hydroxy-phenylethylene glycol-SO4, normetanephrine-SO4, and metanephrine-

Glucuronides of methoxytyramine, normetanephrine, metanephrine, and 3-methoxy-4 hydroxy-phenylethylene glycol are also formed and they may be excreted in the bile. When these glucuronides enter into the circulation, they are eliminated in the urine (Eisenhofer et

Of relevance, the oxidative deamination of catecholamines by MAO leads to another relevant product, hydrogen peroxide (H2O2) which subsequently may be converted into the highly reactive hydroxyl radical (HO●) (Valko et al., 2007), that may cause oxidative stressrelated damage (Carvalho et al., 2001, Duarte et al., 2004, Vaarmann et al., 2010, Costa et al.,

Catecholamines enter the plasma mainly after their leakage from the synaptic cleft to the circulation or after the secretion of catecholamines by the adrenal medulla (Esler et al., 1990, Goldstein et al., 2003). The bulk of NA in the plasma results mainly from sympathetic overflow, while the majority of plasma ADR is formed by adrenal medulla secretion (Esler et al., 1990, Goldstein et al., 2003). The metabolites of NA and ADR also spill into the plasma after intraneuronal metabolism (namely oxidative deamination by MAO) or extraneuronal metabolism (preferentially COMT-methylation), since the majority of the metabolism occurs even before the catecholamines reach the circulation (Goldstein et al., 2003). The most abundant human plasma metabolites are VMA, 3-methoxy-4-hydroxy-phenylethylene glycol and its sulphate metabolite (Goldstein et al., 2003). The main human plasma catechols are the three catecholamines, their precursor, L-3,4-dihydroxyphenylalanine, and their deaminated metabolites, 3,4-dihydroxyphenylacetic acid from dopamine, and DHPG from

In humans, the sympathetic nerves of kidneys and skeletal muscles are the major sources of plasma NA, each contributing with approximately 25% of the total (Esler et al., 1984a, Esler et al., 1984b). The sympathetic innervations of the human heart, skin, gastrointestinal tract, lungs, and liver are responsible for a minor percentage of total plasma NA, but equally important (Esler et al., 1984a, Esler et al., 1984b, Goldstein et al., 1988, Esler and Kaye, 2000). The rate of NA entrance into the arterial plasma ("total body spillover") can be measured and in healthy people averages 0.3 to 0.5 μg/min (Goldstein et al., 2003). Key players in the rate of NA spillover include the rate of NA release, and consequently nerve firing velocity and density, but also regional blood flow, capillary permeability, neuronal NA uptake, and NA metabolism (Esler et al., 1990). These key aspects have to be taken into account when interpreting NA overflow as an index of sympathetic activity. In fact, when comparing the levels of NA release into interstitial fluid to circulation with NA spillover, the NA release into interstitial fluid in kidneys averages 3 times, in skeletal muscle 12 times, and in the heart over 20 times the NA spillover, due to the efficient local neuronal reuptake of NA.

The majority of plasma ADR in physiological conditions results from the stimulation of adrenal medulla (Goldstein et al., 2003); it is assumed that ADR overflow from adrenergic nerves is not very relevant (Esler et al., 1990). The only exception is the heart, which at very

**9.3.1 Levels of catecholamines and their metabolites in the plasma and urine** 

SO4 (Eisenhofer et al., 2004).

NA (Goldstein et al., 2003) (Figure 4).

(Goldstein et al., 2003).

al., 2004).

2011).

In adrenal chromaffin cells, as they contain both MAO and COMT, the deamination and *O*methylation coexist. The COMT present in chromaffin cells is mainly membrane bound COMT while soluble COMT is found in the majority of other tissues (namely liver and kidneys). Membrane bound COMT has a higher affinity for catecholamines than the soluble COMT (Roth, 1992). As a result, in adrenal chromaffin cells, leakage of NA and ADR from storage granules leads to substantial intracellular production of the *O-*methylated metabolites, normetanephrine and metanephrine. In humans, about 93% of circulating metanephrine and between 25% to 40% of circulating normetanephrine result from the catecholamines metabolized within adrenal chromaffin cells (Eisenhofer et al., 2004).

In most cells, the *O*-methylated compounds that contain amine groups undergo further metabolic alterations by MAO. Deamination of 3-methoxytyramine yields HVA and deamination of normetanephrine and metanephrine yields 3-methoxy-4-hydroxyphenylethylene glycol. 3-Methoxy-4-hydroxy-phenylethylene glycol in human plasma is a very important metabolite with multiple sources, including (i) deamination of normetanephrine after its cellular uptake; (ii) *O*-methylation of DHPG after its uptake from the circulation; and (iii) *O*-methylation of DHPG after its uptake from the interstitial fluid but before its entrance into the circulation. Of the previously mentioned sources, the most relevant is the last (Eisenhofer and Finberg, 1994). The fate of circulating 3-methoxy-4 hydroxy-phenylethylene glycol is complex and includes sulfation, glucuronidation, urinary excretion, and specially conversion to methoxy-4-hydroxymandelic acid [generally, called vanillylmandelic acid (VMA)] through the hepatic sequential oxidation of circulating 3 methoxy-4-hydroxy-phenylethylene glycol by alcohol dehydrogenase and aldehyde dehydrogenase (Goldstein et al., 2003). In fact, the major product of urinary excretion and also an important plasma metabolite of adrenergic catecholamines is VMA (Goldstein et al., 2003) (Figure 4).

The formation of normetanephrine occurs after NA extraneuronal uptake and metabolism. Due to the importance of the reuptake and intraneuronal deamination of endogenously released NA, plasma levels of normetanephrine are lower than those of DHPG, despite the similar plasma clearance of these two compounds. The rate of extra adrenal production of normetanephrine, although low, still provides a unique marker of NA extraneuronal metabolism. Accordingly, during MAO-A inhibition, normetanephrine and metanephrine levels increase as COMT becomes more relevant in the metabolism of NA (Eisenhofer and Finberg, 1994).

As stated, adrenomedullary chromaffin cells contain both MAO and COMT and, in agreement with the different affinities between extraneuronal and neuronal uptake, ADR is less metabolized by MAO than NA, but it is a better substrate for COMT (Paiva and Guimarães, 1978, Eisenhofer and Finberg, 1994). Because of these differences, extraneuronal uptake and *O*-methylation clear more circulating ADR than NA (Axelrod et al., 1959, Eisenhofer, 2001, Goldstein et al., 2003) and metanephrine is the major metabolite of ADR (Axelrod et al., 1959, Goldstein et al., 2003).

In cells that contain monoamine-preferring phenolsulfotransferase, the non-acid metabolites, methoxytyramine, normetanephrine, metanephrine, and 3-methoxy-4-hydroxyphenylethylene glycol undergo extensive sulphate-conjugation. The urinary metabolites, resulting from monoamine-preferring phenolsulfotransferase metabolization are usually 3-

In adrenal chromaffin cells, as they contain both MAO and COMT, the deamination and *O*methylation coexist. The COMT present in chromaffin cells is mainly membrane bound COMT while soluble COMT is found in the majority of other tissues (namely liver and kidneys). Membrane bound COMT has a higher affinity for catecholamines than the soluble COMT (Roth, 1992). As a result, in adrenal chromaffin cells, leakage of NA and ADR from storage granules leads to substantial intracellular production of the *O-*methylated metabolites, normetanephrine and metanephrine. In humans, about 93% of circulating metanephrine and between 25% to 40% of circulating normetanephrine result from the

catecholamines metabolized within adrenal chromaffin cells (Eisenhofer et al., 2004).

2003) (Figure 4).

Finberg, 1994).

(Axelrod et al., 1959, Goldstein et al., 2003).

In most cells, the *O*-methylated compounds that contain amine groups undergo further metabolic alterations by MAO. Deamination of 3-methoxytyramine yields HVA and deamination of normetanephrine and metanephrine yields 3-methoxy-4-hydroxyphenylethylene glycol. 3-Methoxy-4-hydroxy-phenylethylene glycol in human plasma is a very important metabolite with multiple sources, including (i) deamination of normetanephrine after its cellular uptake; (ii) *O*-methylation of DHPG after its uptake from the circulation; and (iii) *O*-methylation of DHPG after its uptake from the interstitial fluid but before its entrance into the circulation. Of the previously mentioned sources, the most relevant is the last (Eisenhofer and Finberg, 1994). The fate of circulating 3-methoxy-4 hydroxy-phenylethylene glycol is complex and includes sulfation, glucuronidation, urinary excretion, and specially conversion to methoxy-4-hydroxymandelic acid [generally, called vanillylmandelic acid (VMA)] through the hepatic sequential oxidation of circulating 3 methoxy-4-hydroxy-phenylethylene glycol by alcohol dehydrogenase and aldehyde dehydrogenase (Goldstein et al., 2003). In fact, the major product of urinary excretion and also an important plasma metabolite of adrenergic catecholamines is VMA (Goldstein et al.,

The formation of normetanephrine occurs after NA extraneuronal uptake and metabolism. Due to the importance of the reuptake and intraneuronal deamination of endogenously released NA, plasma levels of normetanephrine are lower than those of DHPG, despite the similar plasma clearance of these two compounds. The rate of extra adrenal production of normetanephrine, although low, still provides a unique marker of NA extraneuronal metabolism. Accordingly, during MAO-A inhibition, normetanephrine and metanephrine levels increase as COMT becomes more relevant in the metabolism of NA (Eisenhofer and

As stated, adrenomedullary chromaffin cells contain both MAO and COMT and, in agreement with the different affinities between extraneuronal and neuronal uptake, ADR is less metabolized by MAO than NA, but it is a better substrate for COMT (Paiva and Guimarães, 1978, Eisenhofer and Finberg, 1994). Because of these differences, extraneuronal uptake and *O*-methylation clear more circulating ADR than NA (Axelrod et al., 1959, Eisenhofer, 2001, Goldstein et al., 2003) and metanephrine is the major metabolite of ADR

In cells that contain monoamine-preferring phenolsulfotransferase, the non-acid metabolites, methoxytyramine, normetanephrine, metanephrine, and 3-methoxy-4-hydroxyphenylethylene glycol undergo extensive sulphate-conjugation. The urinary metabolites, resulting from monoamine-preferring phenolsulfotransferase metabolization are usually 3methoxy-4-hydroxy-phenylethylene glycol-SO4, normetanephrine-SO4, and metanephrine-SO4 (Eisenhofer et al., 2004).

Glucuronides of methoxytyramine, normetanephrine, metanephrine, and 3-methoxy-4 hydroxy-phenylethylene glycol are also formed and they may be excreted in the bile. When these glucuronides enter into the circulation, they are eliminated in the urine (Eisenhofer et al., 2004).

Of relevance, the oxidative deamination of catecholamines by MAO leads to another relevant product, hydrogen peroxide (H2O2) which subsequently may be converted into the highly reactive hydroxyl radical (HO●) (Valko et al., 2007), that may cause oxidative stressrelated damage (Carvalho et al., 2001, Duarte et al., 2004, Vaarmann et al., 2010, Costa et al., 2011).

#### **9.3.1 Levels of catecholamines and their metabolites in the plasma and urine**

Catecholamines enter the plasma mainly after their leakage from the synaptic cleft to the circulation or after the secretion of catecholamines by the adrenal medulla (Esler et al., 1990, Goldstein et al., 2003). The bulk of NA in the plasma results mainly from sympathetic overflow, while the majority of plasma ADR is formed by adrenal medulla secretion (Esler et al., 1990, Goldstein et al., 2003). The metabolites of NA and ADR also spill into the plasma after intraneuronal metabolism (namely oxidative deamination by MAO) or extraneuronal metabolism (preferentially COMT-methylation), since the majority of the metabolism occurs even before the catecholamines reach the circulation (Goldstein et al., 2003). The most abundant human plasma metabolites are VMA, 3-methoxy-4-hydroxy-phenylethylene glycol and its sulphate metabolite (Goldstein et al., 2003). The main human plasma catechols are the three catecholamines, their precursor, L-3,4-dihydroxyphenylalanine, and their deaminated metabolites, 3,4-dihydroxyphenylacetic acid from dopamine, and DHPG from NA (Goldstein et al., 2003) (Figure 4).

In humans, the sympathetic nerves of kidneys and skeletal muscles are the major sources of plasma NA, each contributing with approximately 25% of the total (Esler et al., 1984a, Esler et al., 1984b). The sympathetic innervations of the human heart, skin, gastrointestinal tract, lungs, and liver are responsible for a minor percentage of total plasma NA, but equally important (Esler et al., 1984a, Esler et al., 1984b, Goldstein et al., 1988, Esler and Kaye, 2000).

The rate of NA entrance into the arterial plasma ("total body spillover") can be measured and in healthy people averages 0.3 to 0.5 μg/min (Goldstein et al., 2003). Key players in the rate of NA spillover include the rate of NA release, and consequently nerve firing velocity and density, but also regional blood flow, capillary permeability, neuronal NA uptake, and NA metabolism (Esler et al., 1990). These key aspects have to be taken into account when interpreting NA overflow as an index of sympathetic activity. In fact, when comparing the levels of NA release into interstitial fluid to circulation with NA spillover, the NA release into interstitial fluid in kidneys averages 3 times, in skeletal muscle 12 times, and in the heart over 20 times the NA spillover, due to the efficient local neuronal reuptake of NA. (Goldstein et al., 2003).

The majority of plasma ADR in physiological conditions results from the stimulation of adrenal medulla (Goldstein et al., 2003); it is assumed that ADR overflow from adrenergic nerves is not very relevant (Esler et al., 1990). The only exception is the heart, which at very

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 21

L-3,4-dihydroxyphenylalanine is the precursor of catecholamines and the product of the rate-limiting step of catecholamine synthesis. L-3,4-dihydroxyphenylalanine therefore occupies a crucial role in the catecholaminergic system. In humans, plasma levels of L-3,4 dihydroxyphenylalanine exceed those of NA about 10-fold, reaching 8.9 nM (Goldstein et

Some catecholamine storage vesicle elements are released during exocytosis like chromogranin A, DhB, and neuropeptide Y. Their plasma values can be used as an index of

All catecholamines are ultimately excreted in urine, either in their native form or as metabolites (Esler et al., 1990, Goldstein et al., 2003). Nevertheless, in urine, only a small fraction of catecholamines is present in their unaltered form (Esler et al., 1990) and NA urinary excretion represents only 1-2% of the total NA synthesized. VMA is the major catecholamine metabolite excreted in the urine and 33 μM of VMA can be eliminated each 24 h (Gerlo and Sevens, 1994). The sulphate derivatives of 3-methoxy-4-hydroxyphenylethylene glycol, normetanephrine, and metanephrine are the bulk of the other urinary metabolites (Eisenhofer et al., 2004). The use of urinary excretion levels of catecholamines and/or of their metabolites to estimate total body catecholamine turnover and plasma levels has to be very cautious. Urinary excretion depends on renal blood flow

The metabolization of catecholamines is generally attained either by deamination via MAO or *O*-methylation by COMT. However, the chemical alteration of catecholamines by other enzymes or through oxidative processes is also a probable pathway to the chemical alteration of the transmitters (Richter, 1940)(Figure 5). When the enzymes dealing with the catabolism of catecholamines are unable to cope efficiently, their levels rise and catecholamines can undergo oxidation. The oxidation rate is faster under enzymatic or metal catalysis (Heacock, 1959, Bindoli et al., 1992, Foppoli et al., 1997), in the presence of the superoxide anion (O2•**–**) or high pH (West, 1947, Spencer et al., 1995, Costa et al., 2007). Although at physiological pH, the oxidation of catecholamines seems to occur very slowly, it has been found to occur *in vivo* namely in the septic shock (Macarthur et al., 2000). The oxidation of catecholamines ultimately produces a family of indole semiquinonic/quinonic species usually termed catecholaminochromes because of their orange-reddish colour (Heacock and Mahon, 1958). The oxidation of aqueous extracts of mammal suprarenal capsules was first reported by Vulpian (Vulpian, 1856). The oxidation products of ADR were crucial for the indirect quantification of ADR for many years, since adrenolutin and adrenochrome are easily detected by UV/VIS or by fluorometric methods (Annrsten et al., 1949, Fischer and Bacq, 1950, Ludemann et al., 1955, Remião et al., 2003, Ochs et al., 2004). Generally speaking, catecholamines may be oxidized to an unstable *o-*semiquinone that, after deprotonation and loss of a second electron, gives rise to the corresponding *o-*quinone. For ADR, at physiological pH, partial deprotonation of the amine group of the side chain leads to an irreversible 1,4-intramolecular cyclization, a reaction that occurs through nucleophilic attack of the nitrogen atom at position 6 of the quinone ring, to form "leucoadrenochrome"; leucoadrenochrome is subsequently oxidized to form adrenochrome

and renal function, thus it has some inter-individual variations (Fluck, 1972).

the neuroendrocrine system activation (Esler et al., 1990).

**9.4 Another pathway to degrade catecholamines** 

al., 2003).

high stimulation rates can contribute significantly for ADR spillover into the plasma (Peronnet et al., 1988, Johansson et al., 1997).

Plasma levels of ADR in healthy volunteers at rest are as low as 30 pM, while NA reaches 1 nM (Wheatley et al., 1985, Goldstein et al., 2003). Spillover of ADR to arterial plasma in healthy resting humans is typically 30-100 pg/mL, while it can reach 200-600 ng/mL for NA (Esler et al., 1990). Any alteration in the metabolism of catecholamines or disruption of their transport mechanisms might lead to abnormal high concentrations of these substances (Lameris et al., 2000). Elevated levels of circulating and interstitial catecholamines are found in arrhythmias, myocardial necrosis (Lameris et al., 2000, Behonick et al., 2001), heart failure, (Esler and Kaye, 2000), myocardial ischemia (Lameris et al., 2000, Akiyama and Yamazaki, 2001, Killingsworth et al., 2004, Kuroko et al., 2007), exercise (Kjaer, 1998), pheochromocytoma (Gerlo and Sevens, 1994), hypoglycaemia, haemorrhagic hypotension, circulatory collapse, distress (Goldstein et al., 2003), and cirrhosis (Esler et al., 1990). The administration of amphetamines can also lead to peaks of plasmatic levels of biogenic catecholamines. In fact, after high doses of *d*-amphetamine in rats, levels of plasmatic ADR and NA reached 146 nM and 418 nM, respectively (Carvalho et al., 1997).

The levels of urinary and plasma catecholamine or metabolites can also indicate a higher sympathetic overflow or metabolism impairment (Esler et al., 1990, Brunner et al., 1993, Lenders et al., 1996, Behonick et al., 2001, Goldstein, 2003). In fact, the measurement of DHPG gives important information of NA sympathetic nerve neuronal uptake and turnover (Esler et al., 1990, Goldstein et al., 2003). The DHPG values reflect the sum of vesicular leakage, NA deamination (which is mainly neuronal) and reuptake (Eisenhofer et al., 1992a, Goldstein et al., 2003). DHPG plasma levels can reach 4.7 nM in healthy volunteers (Goldstein et al., 2003).

Formation of normetanephrine in the body occurs after extraneuronal uptake and metabolism of NA in the sympathetic terminals, as well as from the *O*-methylation of NA within the adrenal medulla by COMT. Because of the high importance of reuptake and intraneuronal deamination of NA, plasma levels of normetanephrine (~0.3 nM) are significantly lower than those of DHPG (Goldstein et al., 2003).

*O*-Methylation is the main metabolic pathway of ADR in man (Labrosse et al., 1958). Metanephrine constitutes a major metabolite of ADR before its release into the extracellular fluid (Labrosse et al., 1958, Axelrod et al., 1959). Plasma metanephrine levels are roughly the same as plasma normetanephrine levels, although the levels of plasma NA are about 5- to 10-fold higher than the levels found for ADR. The levels of metanephrine result from the high rate of production of ADR in adrenomedullary chromaffin cells, the metabolism of adrenomedullary catecholamines by COMT (Goldstein et al., 2003), and the relatively high affinity of COMT for circulating ADR (Paiva and Guimarães, 1978, Eisenhofer and Finberg, 1994).

As already stated, 3-methoxy-4-hydroxy-phenylethylene glycol in human plasma has multiple sources, the main being *O*-methylation of DHPG after its uptake from the interstitial fluid but before its entrance into the circulation. The metabolic fate of the circulating 3-methoxy-4-hydroxy-phenylethylene glycol is complex and includes sulfation, glucuronidation, and specially conversion to VMA (Goldstein et al., 2003). 3-Methoxy-4 hydroxy-phenylethylene glycol and VMA constitute the major non sulphate catecholamine metabolites, reaching plasmatic values of 30 and 20 nM, respectively (Goldstein et al., 2003).

high stimulation rates can contribute significantly for ADR spillover into the plasma

Plasma levels of ADR in healthy volunteers at rest are as low as 30 pM, while NA reaches 1 nM (Wheatley et al., 1985, Goldstein et al., 2003). Spillover of ADR to arterial plasma in healthy resting humans is typically 30-100 pg/mL, while it can reach 200-600 ng/mL for NA (Esler et al., 1990). Any alteration in the metabolism of catecholamines or disruption of their transport mechanisms might lead to abnormal high concentrations of these substances (Lameris et al., 2000). Elevated levels of circulating and interstitial catecholamines are found in arrhythmias, myocardial necrosis (Lameris et al., 2000, Behonick et al., 2001), heart failure, (Esler and Kaye, 2000), myocardial ischemia (Lameris et al., 2000, Akiyama and Yamazaki, 2001, Killingsworth et al., 2004, Kuroko et al., 2007), exercise (Kjaer, 1998), pheochromocytoma (Gerlo and Sevens, 1994), hypoglycaemia, haemorrhagic hypotension, circulatory collapse, distress (Goldstein et al., 2003), and cirrhosis (Esler et al., 1990). The administration of amphetamines can also lead to peaks of plasmatic levels of biogenic catecholamines. In fact, after high doses of *d*-amphetamine in rats, levels of plasmatic ADR

The levels of urinary and plasma catecholamine or metabolites can also indicate a higher sympathetic overflow or metabolism impairment (Esler et al., 1990, Brunner et al., 1993, Lenders et al., 1996, Behonick et al., 2001, Goldstein, 2003). In fact, the measurement of DHPG gives important information of NA sympathetic nerve neuronal uptake and turnover (Esler et al., 1990, Goldstein et al., 2003). The DHPG values reflect the sum of vesicular leakage, NA deamination (which is mainly neuronal) and reuptake (Eisenhofer et al., 1992a, Goldstein et al., 2003). DHPG plasma levels can reach 4.7 nM in healthy volunteers

Formation of normetanephrine in the body occurs after extraneuronal uptake and metabolism of NA in the sympathetic terminals, as well as from the *O*-methylation of NA within the adrenal medulla by COMT. Because of the high importance of reuptake and intraneuronal deamination of NA, plasma levels of normetanephrine (~0.3 nM) are

*O*-Methylation is the main metabolic pathway of ADR in man (Labrosse et al., 1958). Metanephrine constitutes a major metabolite of ADR before its release into the extracellular fluid (Labrosse et al., 1958, Axelrod et al., 1959). Plasma metanephrine levels are roughly the same as plasma normetanephrine levels, although the levels of plasma NA are about 5- to 10-fold higher than the levels found for ADR. The levels of metanephrine result from the high rate of production of ADR in adrenomedullary chromaffin cells, the metabolism of adrenomedullary catecholamines by COMT (Goldstein et al., 2003), and the relatively high affinity of COMT for circulating ADR

As already stated, 3-methoxy-4-hydroxy-phenylethylene glycol in human plasma has multiple sources, the main being *O*-methylation of DHPG after its uptake from the interstitial fluid but before its entrance into the circulation. The metabolic fate of the circulating 3-methoxy-4-hydroxy-phenylethylene glycol is complex and includes sulfation, glucuronidation, and specially conversion to VMA (Goldstein et al., 2003). 3-Methoxy-4 hydroxy-phenylethylene glycol and VMA constitute the major non sulphate catecholamine metabolites, reaching plasmatic values of 30 and 20 nM, respectively (Goldstein et al., 2003).

and NA reached 146 nM and 418 nM, respectively (Carvalho et al., 1997).

significantly lower than those of DHPG (Goldstein et al., 2003).

(Paiva and Guimarães, 1978, Eisenhofer and Finberg, 1994).

(Peronnet et al., 1988, Johansson et al., 1997).

(Goldstein et al., 2003).

L-3,4-dihydroxyphenylalanine is the precursor of catecholamines and the product of the rate-limiting step of catecholamine synthesis. L-3,4-dihydroxyphenylalanine therefore occupies a crucial role in the catecholaminergic system. In humans, plasma levels of L-3,4 dihydroxyphenylalanine exceed those of NA about 10-fold, reaching 8.9 nM (Goldstein et al., 2003).

Some catecholamine storage vesicle elements are released during exocytosis like chromogranin A, DhB, and neuropeptide Y. Their plasma values can be used as an index of the neuroendrocrine system activation (Esler et al., 1990).

All catecholamines are ultimately excreted in urine, either in their native form or as metabolites (Esler et al., 1990, Goldstein et al., 2003). Nevertheless, in urine, only a small fraction of catecholamines is present in their unaltered form (Esler et al., 1990) and NA urinary excretion represents only 1-2% of the total NA synthesized. VMA is the major catecholamine metabolite excreted in the urine and 33 μM of VMA can be eliminated each 24 h (Gerlo and Sevens, 1994). The sulphate derivatives of 3-methoxy-4-hydroxyphenylethylene glycol, normetanephrine, and metanephrine are the bulk of the other urinary metabolites (Eisenhofer et al., 2004). The use of urinary excretion levels of catecholamines and/or of their metabolites to estimate total body catecholamine turnover and plasma levels has to be very cautious. Urinary excretion depends on renal blood flow and renal function, thus it has some inter-individual variations (Fluck, 1972).

#### **9.4 Another pathway to degrade catecholamines**

The metabolization of catecholamines is generally attained either by deamination via MAO or *O*-methylation by COMT. However, the chemical alteration of catecholamines by other enzymes or through oxidative processes is also a probable pathway to the chemical alteration of the transmitters (Richter, 1940)(Figure 5). When the enzymes dealing with the catabolism of catecholamines are unable to cope efficiently, their levels rise and catecholamines can undergo oxidation. The oxidation rate is faster under enzymatic or metal catalysis (Heacock, 1959, Bindoli et al., 1992, Foppoli et al., 1997), in the presence of the superoxide anion (O2•**–**) or high pH (West, 1947, Spencer et al., 1995, Costa et al., 2007). Although at physiological pH, the oxidation of catecholamines seems to occur very slowly, it has been found to occur *in vivo* namely in the septic shock (Macarthur et al., 2000). The oxidation of catecholamines ultimately produces a family of indole semiquinonic/quinonic species usually termed catecholaminochromes because of their orange-reddish colour (Heacock and Mahon, 1958). The oxidation of aqueous extracts of mammal suprarenal capsules was first reported by Vulpian (Vulpian, 1856). The oxidation products of ADR were crucial for the indirect quantification of ADR for many years, since adrenolutin and adrenochrome are easily detected by UV/VIS or by fluorometric methods (Annrsten et al., 1949, Fischer and Bacq, 1950, Ludemann et al., 1955, Remião et al., 2003, Ochs et al., 2004).

Generally speaking, catecholamines may be oxidized to an unstable *o-*semiquinone that, after deprotonation and loss of a second electron, gives rise to the corresponding *o-*quinone. For ADR, at physiological pH, partial deprotonation of the amine group of the side chain leads to an irreversible 1,4-intramolecular cyclization, a reaction that occurs through nucleophilic attack of the nitrogen atom at position 6 of the quinone ring, to form "leucoadrenochrome"; leucoadrenochrome is subsequently oxidized to form adrenochrome

Adrenaline and Noradrenaline: Partners and Actors in the Same Play 23

groups can be involved (Spencer et al., 1998, Bindoli et al., 1999, Spencer et al., 2002, Costa et

At physiological pH, the oxidation pathway of biogenic catecholamines is similar, as they share similar intermediates, however their stability is fairly different (Rupp et al., 1994). The rate of internal cyclization determines the probability of nucleophilic attack to the quinone intermediates (Rupp et al., 1994, Spencer et al., 1995, Alhasan and Njus, 2008). Thus, if the internal cyclization rate is low, the quinone is likely to be attacked by external nucleophilic groups such as sulfhydryl (-SH), hydroxyl (-OH), and amine (-NH2) groups. The slowest cyclization rate is observed for dopamine, while ADR has the higher cyclization rate, 140 times higher than NA. Thus, the probability of external nucleophilic attack to occur is as following: dopamine > NA > ADR (Rupp et al., 1994). In fact, dopamine oxidized forms were found bound to proteins, as well as to other nucleophilic molecules, as cysteine or glutathione (Fornstedt et al., 1990, Patel et al., 1991, Segura-Aguilar et al., 1997, Byington, 1998, Spencer et al., 1998, Spencer et al., 2002, Miyazaki et al., 2006). Pathognomonic significance has been given to these products, namely in Parkinson's disease (Spencer et al., 1995, Shen et al., 1996, Spencer et al., 1998). As stated, the fastest cyclization rate occurs in ADR (Bindoli et al., 1992); however, GSH adducts and quinoproteins of ADR have been found in cells, thus showing that ADR quinone can also undergo attack from cellular nucleophiles (Costa et al., 2007, Costa et al., 2009a, Costa et al., 2009c). These changes can

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

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 β-

material, we may speak of it as the receptive substance (…)" (Langley, 1905).

al., 2007, Costa et al., 2009a).

ultimately cause cellular injury (Costa et al., 2011).

**10.1 Historic introduction and background** 

**10. Adrenergic receptors** 

adrenoceptors (Black et al., 1964).

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

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

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

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

Catecholamine

groups can be involved (Spencer et al., 1998, Bindoli et al., 1999, Spencer et al., 2002, Costa et al., 2007, Costa et al., 2009a).

At physiological pH, the oxidation pathway of biogenic catecholamines is similar, as they share similar intermediates, however their stability is fairly different (Rupp et al., 1994). The rate of internal cyclization determines the probability of nucleophilic attack to the quinone intermediates (Rupp et al., 1994, Spencer et al., 1995, Alhasan and Njus, 2008). Thus, if the internal cyclization rate is low, the quinone is likely to be attacked by external nucleophilic groups such as sulfhydryl (-SH), hydroxyl (-OH), and amine (-NH2) groups. The slowest cyclization rate is observed for dopamine, while ADR has the higher cyclization rate, 140 times higher than NA. Thus, the probability of external nucleophilic attack to occur is as following: dopamine > NA > ADR (Rupp et al., 1994). In fact, dopamine oxidized forms were found bound to proteins, as well as to other nucleophilic molecules, as cysteine or glutathione (Fornstedt et al., 1990, Patel et al., 1991, Segura-Aguilar et al., 1997, Byington, 1998, Spencer et al., 1998, Spencer et al., 2002, Miyazaki et al., 2006). Pathognomonic significance has been given to these products, namely in Parkinson's disease (Spencer et al., 1995, Shen et al., 1996, Spencer et al., 1998). As stated, the fastest cyclization rate occurs in ADR (Bindoli et al., 1992); however, GSH adducts and quinoproteins of ADR have been found in cells, thus showing that ADR quinone can also undergo attack from cellular nucleophiles (Costa et al., 2007, Costa et al., 2009a, Costa et al., 2009c). These changes can ultimately cause cellular injury (Costa et al., 2011).
