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Can catecholamines degrade back into tyrosine, or, is synthesis irreversible? (in human body)

Can catecholamines degrade back into tyrosine, or, is synthesis irreversible? (in human body)


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Catecholamines like dopamine, noradrenaline and adrenaline are broken down with enzymes that catalyze the reaction. Can they degrade back into tyrosine (a conditionally essential amino acid), or is the synthesis of catecholamines like adrenaline irreversible? (in context human body, but, broadest possible phylogenetic or biochemical context welcome in answer… )


A glance at the relevant BioCyc entry tells us that each reaction in the catecholamine biosynthesis pathway is irreversible. The standard free energy change of each reaction-again from BioCyc-is given below.

egin{array} {|r|r|} hline ext{Reaction} & Delta _r G' ^{circ} ext{ in kcal mol}^{-1} hline ext{Tyrosine hydroxylation} & -93.0 hline ext{Decarboxylation of DOPA} & -3.09 hline ext{Hydroxylation of dopamine} & -67.9 hline ext{Norepinephrine to epinephrine} & -10.5 hline end{array}

Note that all the free energy changes are negative, i.e. all reactions are irreversible under physiologic circumstances. (For the last one, there is a simple explanation: S-adenosylmethionine is a powerful methylator [1].)

Another aspect is worth a mention. Under physiologic conditions, the reversibility of a reaction depends not only on the free energy changes involved, but also on what happens to the reactants and products. A large supply of reactants, or rapid removal of reaction products, can make the reaction physiologically irreversible. Applying this principle to the catecholamine biosynthesis pathway:

  1. A steady supply of oxygen is available from the atmosphere-this drives the tyrosine and dopamine hydroxylation reactions in the forward direction.

  2. The dihydrobiopterin produced by tyrosine hydroxylase is scavenged by reduction to tetrahydrobiopterin, which further takes part in the tyrosine hydroxylase reaction [1].

  3. The carbon dioxide produced by DOPA decarboxylase is exhaled out.

This further adds to irreversibility.

Of course, as mentioned by @user1136 in the comments, it is theoretically possible to have a "reverse" pathway by coupling to ATP hydrolysis. However, I could not find any such pathway in database searches.

Summary: To the best of my knowledge, humans cannot convert catecholamines back to tyrosine.


Reference

  1. Nelson DL, Cox MM. Lehninger principles of biochemistry. 6th ed. New York: WH Freeman and Company; c2013. Chapter 18, Amino acid oxidation and the production of urea; p 695-730.

I do believe it's irreversible. When catecholamines have done their duty, they are degraded by COMT and MAO into products that are excreted in the urine.

I'll make an example out drugs that are MAO inhibitors. The enzyme MAO participates in the degradation of dopamine, NE, and Epi. MAO-A inhibitors are non-selective and used for depression/anxiety because their inhibition will increase levels of dopamine and NE (this might help show that there is no reverse reaction, because we don't see an increase in tyrosine). MAO-B inhibitors are dopamine specific, and therefore used in parkinsons which is a disease of low dopamine.

Hope this helps!


Catecholamine

Catecholamines (Norepinephrine, Dopamine) and Related Compounds

The catecholamines , norepinephrine (NE noradrenaline), and dopamine (DA), are formed from the amino acid tyrosine, which itself is derived from the amino acid phenylalanine. Cell bodies of the noradrenergic system are primarily located in the locus coeruleus. High concentrations of NE are also found in the hypothalamus. Dopamine is present in high levels in the neostriatum, nucleus accumbens, and olfactory tubercules. In the CNS, the major metabolite of NE is 3-methoxy-4-hydroxyphenylglycol (MHPG). In the peripheral sympathetic nervous system, vanillylmandelic acid (VMA) is the primary metabolite of NE. In the human brain, the primary metabolite of dopamine is homovanillic acid (HVA), with a smaller amount of 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine formed. Free MHPG diffuses from the brain into the CSF and general circulation, so estimates of its concentration in the CSF are thought to reflect CNS noradrenergic neuronal activity. A proportion of CSF MHPG derives from the plasma, because free MHPG diffuses readily through membranes, so correction for the plasma contribution must be made. Plasma and urinary free MHPG may be useful indices of total body NE metabolism, but are not valid indices of brain NE metabolism, as the brain accounts for only approximately 30% of the total body production of MHPG. CSF HVA levels are taken as an index of DA metabolism in the CNS.

The few studies that have been done investigating CSF MHPG in violent offenders suggest reduced levels of this neurochemical are associated with violent behavior. Impulsive arsonists (relative to habitually violent offenders and nonviolent controls) and alcoholic violent offenders and impulsive fire-setters with histories of violent suicide attempts (relative to those without) have low CSF MHPG. Low CSF MHPG also predicted recidivism in these individuals. Two other studies have found no differences in CSF NE or MHPG concentrations between groups of impulsive versus nonimpulsive violent offenders, so the relationship between violence and CSF MHPG remains to be confirmed.

The noradrenergic system contributes to the regulation of arousal and responsiveness to the environment. Increased locus coeruleus activity has been associated with reactivity to novel and particularly threatening stimuli, decreased activity with self-restitutive or vegetative activity such as eating, self-grooming, and sleeping. The few studies that did find lower CSF MHPG in arsonists and impulsive violent offenders may imply reduced arousal and reactivity in these individuals. This is consistent with psychophysiological studies of psychopaths showing reductions in sympathetic nervous system arousal (e.g., skin conductance activity) compared to nonpsychopaths.

The relatively few studies investigating NE functioning in personality-disordered individuals suggest a positive correlation with aggression. CSF MHPG levels were positively correlated with self-reported aggression in personality-disordered military personnel (although the variance in aggression explained was low after controlling for CSF 5-HIAA). In another study, growth hormone responses to the alpha-2-adrenergic agonist clonidine were positively correlated with sensation-seeking and risk-taking behaviors, and self-reported lifetime irritability (but not assaultiveness) in both personality-disordered patients and normal controls. (Note that there was no correlation between growth hormone responses and overt aggression or impulsivity in this study.) In another study with personality-disordered individuals, growth hormone responses to clonidine were associated with irritability and verbal hostility.

These results are difficult to reconcile with those found in violent offenders, which suggest that increased NE function is related to aggression and violence. It has been suggested that reduced presynaptic and increased postsynaptic NE function are related to aggression. Increased presynaptic alpha-2 NE receptor activity in the locus coeruleus (as has been suggested by increased locus coeruleus alpha-2 agonist binding in violent, but not nonviolent, suicides victims) may lead to reduced NE outflow (and thus reduced CSF MHPG) and an upregulation of postsynaptic alpha-2 NE receptors, thus increasing growth hormone responses to clonidine. Overall, this might lead to an augmented alpha-2 NE signal in NE pathways in the presence of aversive, provocative stimuli, resulting in a heightened behavioral arousal (fight/flight response). Furthermore, alpha-2 heteroceptors terminating on presynaptic 5-HT neurons may be supersensitive, leading to greater inhibition of 5-HT firing. Such a synthesis is intriguing and provides a number of testable hypotheses.

Less evidence in humans implicates the dopaminergic system in violent behavior. Studies on violent offenders have found no differences in CSF HVA levels in arsonists versus habitually violent offenders and nonviolent controls, convicted violent criminals who committed impulsive versus premeditated crimes, violent offenders and impulsive fire-setters with histories of serious violent suicide attempts compared to those with no such histories, or between impulsive alcoholic violent offenders with antisocial personality disorder, impulsive alcoholic violent offenders with intermittent explosive disorder, nonimpulsive alcoholic violent offenders and healthy volunteers. Violent criminals with antisocial personality disorder had lower levels of CSF HVA compared to those with paranoid or passive-aggressive personality disorders. CSF DA levels and turnover were not different between five XYY assaultive patients and controls. No differences in CSF DOPAC or HVA have been found between convicted violent criminals who committed impulsive versus premeditated crimes.

One study has found that violent alcoholic offenders had slightly higher striatal dopamine reuptake site densities compared to nonviolent alcoholic offenders and nonalcoholic controls, suggesting that violence is associated with increased dopaminergic activity in the brain.

Finally, two studies suggested that violent male offenders may be either higher or lower in their levels of free or conjugated plasma phenylacetic acid, the major metabolite of phenylethylamine following its breakdown by monoamine oxidase (MAO) B, compared to nonviolent offenders. Phenylethylamine, structurally related to amphetamine, has a similar pharmacological response to the latter when administered following pre-treatment with an MAO inhibitor. It is synthesized from the amino acid phenylalanine. It has been posited that increased phenylacetic acid levels in violent offenders may indicate increased phenylethylamine levels, the latter representing a compensatory increase to reduce aggressive tendencies, much the way amphetamine reduces hyperactivity in attention-deficit and hyperactive children.


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Monoamines (also known as "biogenic amines") include three classes of neurotransmitters:

  • Catecholamines
    • Dopamine (DA), norepinephrine (NE, also called noradrenaline) and epinephrine (E, also called adrenaline) make up a class of neurotransmitters named on the basis of the hydroxylated phenol ring termed a catechol nucleus.
    • Serotonin (5-hydroxytryptamine 5-HT) is the principal member of this group of compounds. The name serotonin is derived from the fact that this substance was first isolated from the serum based on its ability to cause an increase in blood pressure. Melatonin, a second indolamine, is restricted to the pineal and is released into the blood stream in a manner that is regulated by the diurnal cycle. Melatonin will not be covered further in this chapter.
    • Histamine has been recognized as a neurotransmitter in the CNS only within the past fifteen years.

    The structure of the monoamine neurotransmitters is shown in Figure 12.1.

    Figure 12.1
    Structure of the monoamine neurotransmitters.

    12.2 Anatomy of Catecholamines

    Catecholamines are neurotransmitters in a sympathetic limb of the autonomic nervous system and in the CNS.

    12.3 Autonomic Nervous Systems

    As shown in Figure 12.2, norepinephrine is a neurotransmitter in postganglionic sympathetic neurons where it acts on smooth muscle to cause either contraction or relaxation, depending on the types of receptors present (see below). DA is a neurotransmitter in autonomic ganglia where it modulates cholinergic transmission and in the kidney, where it produces renal vasodilation and inhibits Na + and H2O reabsorption. Epinephrine and norepinephrine are neurohumoral agents released into the circulation by the adrenal medulla. The ratio of E to NE released is 4 to 1.

    Figure 12.2
    Location of NE and Epi at sympathetic nerve endings and in the adrenal medulla.

    12.4 Central Nervous System

    Generally, the cell bodies of catecholamine neurons are found in clusters in the brain stem or midbrain and project to other regions of the brain and spinal cord. NE, for example, projects to almost every area of the brain. In contrast, DA has a more restricted projection. Epinephrine, which will not be covered in this chapter, has the most restricted distribution.

    Figure 12.3
    Location of DA in the rat CNS

    The major site of DA cell bodies is the midbrain. These clusters of cells give rise to four DA systems shown in Figure 12.3:

    1. Mesostriatal system (blue and red in Figure 12.3),
    2. Mesolimbocortical system (purple in Figure 12.3),
    3. Periventricular system (orange in Figure 12.3), and
    4. Tuberohypophyseal system. (green in Figure 12.3).

    Mesostriatal DA System - The mesostriatal DA system, referred to as the nigrostriatal pathway, is composed of two components, a dorsal mesostriatal pathway and a ventral mesostriatal pathway. These two pathways are important for movement control and reward mechanisms.

    The dorsal mesostriatal pathway (blue) originates in the substantia nigra par compacta and ascends to innervate the corpus striatum (caudate, putamen, and globus pallidus), where it modulates the output of the corpus striatum. The destruction of the nigrostriatal cells in Parkinson's disease produces marked motor deficits.

    The ventral mesostriatal pathway (red) also originates in the substantia nigra and ventral tegmental area, and innervates the nucleus accumbens, olfactory tubercles and medial caudate-putamen. The ventral nigrostriatal pathway plays an important role in positive incentive characteristics of rewarding behaviors and the psychostimulants, as will be discussed further in Chapter 12, Part 10.

    Mesolimbocortical DA System - The Mesolimbocortical DA system (purple) originates in the midbrain and projects to limbic structures (septum, amygdala, hippocampus, olfactory nucleus, and limbic cortex). This DA system is believed to participate in schizophrenia. (See Chapter 12, Part 11) The current hypothesis is that an increase in DA function in the mesolimbic system and a decreased function in the mesocortical DA systems occur in schizophrenia.

    Periventricular DA System - The periventricular DA system (orange in Fig. 12.3) coordinates motivated behavior. These DA cells originate in the periventricular region of hypothalamus and send short axons to several thalamic and hypothalamic nuclei. Collaterals also descend to the intermediolateral cell column of the spinal cord to synapse with sympathetic preganglionic neurons. This dual innervation of hypothalamic and sympathetic preganglionic neurons is believed to integrate the central and autonomic components of motivated behaviors, including behaviors such as sex, thirst and appetite.

    Tuberohypophyseal DA System - The tuberohypophyseal DA system mediates the control of milk production during lactation. The DA cells (green) originate in the periventricular and arcuate nuclei of the hypothalamus and project to the median eminence of the hypothalamus where they release DA into the capillary plexus of the hypophyseal-portal system. DA travels to the anterior pituitary where it inhibits the release of prolactin, the hormone that stimulates milk production in lactating animals.

    12.6 Norepinephrine - Anatomy

    Figure 12.4
    Location of NE in the rat CNS.

    The major site of NE cell bodies is the medulla and pons. The NE cells consist of three main groups shown in Figure 12.4:

    1. locus coeruleus complex (purple and red in Figure 12.4),
    2. lateral tegmental system (blue in Figure 12.4), and
    3. dorsal medullary system (green in Figure 12.4).

    In all three cases the neurons project diffusely to broad regions of the brain where their nerve terminals lack conventional synaptic junctions. Release of transmitter from these cells is described as volume transmission, because NE, once released, is thought to diffuse and influence a number of adjacent cells.

    Locus Coeruleus System - The locus coeruleus (LC-purple and red) is considered the most influential of the cell groups even though it consists of less than 2,000 cells on either side of the midline. This importance is because LC axons project rostrally via the dorsal noradrenergic bundle to innervate nearly the entire telencephalon and diencephalon, as well as dorsally to innervate the cerebellum and caudally to innervate the spinal cord. The nerve fibers are so highly ramified in the terminal fields such that each axon may branch as many as 100,000 times. This pattern of innervation enables the LC to synchronously modulate cellular activity across wide expanses of the cortex.

    Lateral Tegmental System - The axons of the lateral tegmental system (blue) project caudally to the intermediolateral cell column of the spinal cord where they inhibit sympathetic preganglionic cells, and ventrally to the hypothalamus. The joint innervation of the hypothalamus and the intermediolateral column cells is believed to be the basis for NE integration of central and peripheral sympathetic autonomic function.

    Dorsal Medullary System - As a complement to the lateral tegmental system, the dorsal medullary system (green) projects to the nucleus solitarius, as well as to the brain stem nuclei that control cranial parasympathetic function (glossopharyngeal, facial, and trigeminal- nuclei and the dorsal vagal nuclear complex). These NE systems are believed to provide control of the cranial parasympathetic system in a manner analogous to the lateral tegmental system's control of the sympathetic system.

    (Note: there is no figure for epinephrine anatomy)

    Two clusters of epinephrine (E) cells are located in the medullary reticular formation. One cluster of cells in the ventrolateral medulla sends ascending projections to innervate the periaqueductal gray and several hypothalamic and olfactory nuclei. This cluster also sends a descending projection to innervate the sympathetic preganglionic cells of the intermediolateral column in a manner analogous to the NE lateral tegmental system. The second group of cells, located in the dorsomedial medulla near the floor of the fourth ventricle, project to several parasympathetic cranial nerve nuclei (similar to the dorsal medullary NE system, described above). These adrenergic cells are believed to coordinate eating and various visceral functions including the regulation of blood pressure.

    Figure 12.5
    5-HT neuronal pathways in the rat CNS are in two clusters of cells, one in the caudal brain stem (B1-B4) and the other in the rostral brainstem (B5-B9)

    As shown in Figure 12.5, serotonin cells are located in two clusters:

    1. a caudal system in the medulla (B1-B4, green in Figure 12.5)
    2. a rostral system in the midbrain (B5-B9, purple and blue in Figure 12.5).

    Both project widely throughout the CNS.

    Caudal System: The caudal cluster of 5-HT cells (B1-B4) is located close to the midline and project caudally to the spinal cord dorsal and ventral horns as well as the intermediolateral cell column. These pathways are believed to mediate the role of 5-HT in sensory, motor and autonomic functions, respectively.

    Rostral System: The rostral midbrain cluster of cells (B5-B9), (raphe nuclei) are distributed throughout the midbrain. A cluster of cells located medially and another located dorsally provide over 80% of the 5-HT innervation of the forebrain. These cells project to the diencephalon, basal ganglia, limbic system, cortex, mesencephalic gray and inferior and superior colliculi. Some evidence supports the conclusion that the innervation of forebrain structures by serotonergic processes is complementary to that of NE. Another important aspect of 5-HT microanatomy is that two distinct patterns of innervation exist for these medial and dorsal systems. The dorsal system is similar in its anatomy to that of catecholamine neurons with thin diffusely branching axons lacking classic synaptic contacts (volume neurotransmission). The medial system, in contrast, appears to have classical synapses and is characterized by the presence of thick axons with large round nerve endings that make extensive synaptic contacts. These differences imply a marked difference in the physiological function of these two 5-HT systems.

    Other Systems: In addition to the above two pathways, another 5-HT pathway projects partially from one of the rostral nuclei (B5) and partially from two caudal nuclei (B2 & B3, dark green in Figure 12.5) to innervate the cerebellar cortex and deep cerebellar nuclei. There is also a widespread 5-HT projection to structures within the brainstem, including the locus coeruleus, several cranial nuclei, inferior olivary nucleus, and nucleus solitarius.

    Figure 12.6
    Histamine neuronal pathways mapped in the rat CNS using histidine decarboxylase immunoreactivity.

    Histamine cells (HA) are located exclusively in the basal posterior hypothalamus. These cells project extensively throughout the neural axis in a manner analogous to the NE and 5-HT systems. Although HA has not been investigated extensively, based on its diffuse innervation of the CNS and lack of classic synaptic contacts, it is likely that histamine has a broad behavioral and physiological function.

    Histamine is also the major active substance released from mast cells. The presence of mast cells in the blood in the CNS has hindered the analysis of the role of histamine as a neurotransmitter.

    12.10 Introduction to Cell Biology

    The monoamines will be considered as a group in discussing the cell biology of their 1) synthesis, 2) storage and 3) release. Monoamine receptors and termination of action of each monoamine will be considered separately.

    12.11 Cell Biology - Biosynthesis of Monoamines

    All monoamine (MA) neurotransmitters are synthesized from amino acids through a series of enzyme catalyzed reactions in which hydroxylation, decarboxylation and/or methylation convert the precursor amino acid into the active monoamine neurotransmitter.

    Biosynthesis of Dopamine (DA), Norepinephrine (NE), and 5-hydroxytryptamine (5-HT)

    Biosynthesis of all monoamines occurs primarily in the nerve terminal. As shown in Figure 12.7, the first step in the synthesis of catecholamines (DA and NE, as well as E, not shown) is the hydroxylation of the tyrosine to form DOPA. An analogous reaction, the hydroxylation of tryptophan to 5 hydroxytryptophane (5-HTP) is the first step in the biosynthesis of 5-HT. Both tyrosine hydroxylase and tryptophan hydroxylase are the rate-limiting steps in the biosynthetic pathway of the respective monoamines. Both enzymes are mixed function mono-oxygenases requiring molecular oxygen, iron and the cofactor, tetrahydrobiopterin (BH4) for activity. BH4 is converted to BH2 during the hydroxylation and must be regenerated to BH4 in order for monoamine biosynthesis to continue. As shown in Figure 12.7, the enzyme pteridine reductase regenerates the active cofactor. Pteridine reductase is therefore also an essential enzyme in the synthesis of catecholamines. The next step in the biosynthesis of monoamines is the decarboxylation by aromatic amino acid decarboxylase (AADC) to form the corresponding monoamine (Dopamine and 5 hydroxytryptamine 5-HT, respectively). NE is then formed from dopamine through an additional reaction, the hydroxylation of the 2 nd carbon of the DA side chain. This last hydroxylation step occurs within the monoamine storage vesicle (see Figure 12.9a) and is catalyzed by dopamine β hydroxylase.

    Biosynthesis of the DA and NE precursor L-DOPA and the 5-HT precursor 5-HTP through hydroxylation using tyrosine hydroxylase (TH) and tryptophan hydroxylase (TryH.) These intermediates are then decarboxylated by a nonspecific decarboxylase, aromatic amino acid decarboxylase (AADC) to form the respective monoamines. Pteridine reductase regenerates the cofactor BH4.Biosynthesis of the DA and NE precursor L-DOPA and the 5-HT precursor 5-HTP through hydroxylation using tyrosine hydroxylase (TH) and tryptophan hydroxylase (TryH.) These intermediates are then decarboxylated by a nonspecific decarboxylase, aromatic amino acid decarboxylase (AADC) to form the respective monoamines. Pteridine reductase regenerates the cofactor BH4.

    Two additional cofactors are required for the synthesis of monoamines vitamin B6 is necessary as a cofactor for AADC catalyzed decarboxylation. Vitamin C is required as a cofactor for DA conversion to NE in the storage vesicle (see Figure 12.9a).

    Biosynthesis of Epinephrine (E)

    Epinephrine is synthesized in adrenal medulla and CNS by methylation of NE on the amino-terminus (not shown). The enzyme that catalyzes this reaction is phenyl ethanolamine N methyl transferase (PNMT). This enzyme uses S-adenosyl methionine as the methyl donor to methylate norepinephrine to form epinephrine (the nor refers to the lack of the methyl group). PNMT's localization outside the storage vesicle requires that norepinephrine shuttle out of the vesicle to be converted to epinephrine and then back into the storage vesicle for storage and release.

    Biosynthesis of Histamine (HA)

    Figure 12.8
    Biosynthesis of histamine from histidine.

    As shown in Figure 12.8, in contrast to the catecholamines and 5-HT, the biosynthesis of histamine does not require hydroxylation. Histamine is the product of the decarboxylation of the amino acid, histidine, to form the monoamine, histamine, in a single step that is analogous to the decarboxylation of DOPA and 5-HTP. A different enzyme is used to decarboxylate histidine, histidine decarboxylase, as shown in Figure 12.8. This enzyme, like AADC, requires vitamin B6.

    Regulation of Catecholamine Biosynthesis

    The concentration of catecholamines in nerve terminals remains relatively constant despite frequent fluctuations in neuronal activity. This homeostasis is achieved through the regulation of TH activity. TH is phosphorylated and activated by both calcium and cAMP dependent protein kinases. A longer-term regulation of CA synthesis also occurs. This regulation is mediated through altered transcription of TH mRNA and altered TH mRNA stability. Both mechanisms lead to increased levels of TH protein.

    Regulation of Serotonin Biosynthesis

    The level of serotonin is regulated principally by the amount of tryptophan available to serotonergic neurons. This has two important implications for the level of serotonin in the brain. First, because tryptophan is not synthesized in mammals, the level of tryptophan available for serotonin biosynthesis is dependent on diet. Thus, diets high in tryptophan can markedly elevate serotonin levels. Second, because tryptophan is transported across the blood brain barrier by a transport system which also transports certain other amino acids, diets high in these amino acids can reduce the level of serotonin in the brain by competing with tryptophan for transport into the CNS. As will be discussed later, altered serotonin level in the CNS can have marked consequences on behavior.

    Regulation of Histamine Biosynthesis

    Thus far the mechanism for the regulation of histamine biosynthesis is unknown.

    12.12 Storage of Monoamines

    Monoamine neurotransmitters are stored in vesicles that appear dark at the EM level and are thus referred to as dense core vesicles. MA neurotransmitters are stored at a high concentration and are complexed with ATP and several proteins called chromogranins. One of these chromogranins is the enzyme, dopamine β hydroxylase (D β H), that converts DA to NE. As shown in Figure 12.9, MA neurotransmitters are taken into the vesicles by an exchange of H + for the MA. In NE cells DA is taken up and converted to NE by D β H. As described above in the synthesis section, D β H hydroxylates the amino side chain. The uptake of MA neurotransmitters into storage vesicles is inhibited by the drug reserpine.

    An antiporter that exchanges protons for monoamines (MA) mediates storage of monoamines in dense core vesicles. Left: In NE cells, DA is taken up then converted to NE within the vesicle by the enzyme DBH. Right: All other monoamine cells merely store the MA neurotransmitters.

    12.13 Release of Monoamines

    Figure 12.10
    MA release and interaction with both presynaptic and postsynaptic receptors.

    Neuronal activation elicits the release of MA neurotransmitters by a calcium-dependent exocytosis, as described in Lecture 10, under Secretory Mechanism. The vesicular contents are released from the nerve terminal into the extracellular space during secretion. Because there is no classic postsynaptic specialization associated with the majority of MA nerve endings, the released MA neurotransmitters diffuse to postsynaptic cells in the vicinity where they stimulate MA receptors (volume neurotransmission).

    MA neurotransmitters also act on the presynaptic cell, as shown in Figure 12.10 to influence their cell biology in a feed back manner. The interaction with the presynaptic receptors (termed autoreceptors) can both stimulate MA biosynthesis and inhibit the further release of neurotransmitter. Both the pre- and postsynaptic MA receptors are G protein linked, seven trans-membrane receptors. Their structure is similar to the muscarinic receptors discussed in the Lecture 11, Cholinergic Neurotransmission.

    12.14 Properties of Monoamine Receptors

    The vast majority of the MA receptors are seven transmembrane, G-protein coupled receptors (GPCR) that mediate MA action through one of a few mechanisms. These are the same mechanisms employed by other GPCR, such as the muscarinic receptors (Chapter 12, Part 5) and GPC-glutamate receptors (Chapter 13, Part 3). These mechanisms are:

    1. Stimulation or inhibition of adenylyl cyclase (Click here to see mechanism),
    2. Stimulation of PLC β or PLA (Click here to see mechanism), and
    3. Direct action on ion channel (Click here to see mechanism).

    As will be described below, one type of MA receptor, 5-HT3, is unusual in that it is NOT LINKED TO G PROTEIN LINKED RECEPTORS. Instead, 5-HT3 receptors are ligand gated ion channels, similar in structure and function to ionotropic nicotinic cholinergic receptors and glutamate receptors.

    The receptors for NE and E were originally classified based on the observation that some physiological actions were mimicked by the catecholamine analog, isoproterenol, whereas others were not. This observation led to the convention that actions that could be mimicked by isoproterenol were classified as mediated by β -receptors. Those actions that were not mimicked by isoproterenol were classified as mediated by α -receptors.

    12.16 Relationship Between Peripheral NE and E Receptor Type, Location and Effector Mechanism

    This classification has since been extended to include subclasses of α and β receptors based on the capacity of drugs to selectively activate (or block) specific physiological responses to NE and E. The molecular cloning of mRNAs for distinct subclasses of NE and E receptors also aided in the classification of receptors. Tables I, II and III summarize autonomic and CNS NE and E receptor types, their location and their physiological action. Noteworthy is the fact that most α receptor responses are excitatory, while most β responses are inhibitory (although some exceptions exist, e.g. cardiac muscle). Also, the α receptor is invariably linked to IP3 production, whereas the β receptor is associated with increased levels of cAMP.

    Table I
    Relationship Between Peripheral NE and E Receptor Type, Location, and Effector Mechanism
    Class Location Synaptic Action Linked to:
    α Uterine muscle Contraction IP3 production
    α Blood vessels Constriction IP3 production
    α Bladder Contraction IP3 production
    α Spleen Contraction IP3 production
    α Iris Pupil dilation ?
    β 1 Heart Increased rate and force of contraction Increased cAMP
    β 2 Blood vessels Relaxation Increased cAMP
    β 2 Bronchial muscle Relaxation Increased cAMP
    β 2 Bladder Relaxation Increased cAMP
    β 2 Spleen Relaxation Increased cAMP
    β 3 Fat cells Lipolysis Increased cAMP

    12.17 Relationship Between CNS NE Receptor Type and Effector Mechanism

    The distribution of NE receptors in the CNS is complex and not yet well resolved. Generally, both α and β receptors are believed to be modulators of the actions of other neurotransmitters. As summarized in Table II, α 1 receptors are often excitatory, acting via IP3. In contrast, α 2 receptors are inhibitory acting via decreased levels of cAMP. β receptors are inhibitory and act through increased levels of cAMP (TABLE II). The anatomical location of the specific receptor subtypes is not yet clearly delineated.

    Table II
    Relationship Between CNS NE Receptor Type and Effector Mechanism
    Class Synaptic Action Signaling Mechanism
    α 1 Slow depolarization IP3 production
    α 2 Slow hyperpolarization Decreased cAMP
    β 1 Decreased excitability Increased cAMP
    β 2 Decreased excitability Increased cAMP

    In the CNS, dopamine receptors, designated by the letter D, are grouped into two large families based on cDNA-derived structural similarities, synaptic action and signaling mechanism (TABLE III). The D1 family (D1 and D5) increases cAMP level, and has a positive influence on the excitability of its target cell. The D2 family (D2, D3, and D4) decreases cAMP level and decreases the excitability of the target cell. As shown in Table III the two families of receptors appear to have similar anatomical distributions. However, this may be misleading. Future research will probably show that the location of the receptors is on distinct postsynaptic cells or on presynaptic versus postsynaptic sites.

    12.19 Relationship Between CNS Dopamine Receptor Type, Location, and Effector Mechanism

    Table III
    Class Location Synaptic Action Signaling Mechanism
    D1 family
    (D1, D5)
    Caudate -putamen, nucleus accumbens, olfactory tubercles, hippocampus, hypothalamus Increased excitability Increased cAMP
    D2 family
    (D2, D3, D4)
    Caudate -putamen, nucleus accumbens, olfactory tubercles, frontal cortex, diencephalon, brain stem Decreased excitability Decreased cAMP

    All but one of the 5-HT receptors belongs to the G protein coupled receptor superfamily. The one exception is the 5-HT3 receptor, which is a ligand gated ion channel. As is apparent from the summary in Table IV, 5-HT mediated actions occur through the same types of second messenger mechanisms as cholinergic and catecholamine G protein linked receptors.

    Two classes of 5-HT receptors, 5-HT1B and 5-HT1D, appear to predominantly act as autoreceptors to modulate the synthesis and release of 5-HT from the presynaptic terminal of serotonergic neurons. Other receptor types lead to an increase in the excitability of the target cell (5-HT2 and 5-HT4), while still others (5-HT1) decrease excitability. Interestingly, receptors that mediate increased excitability do so through at least three mechanisms, PLC β stimulation, stimulation of adenylyl cyclase or the direct interaction of 5-HT with the ion channel to depolarize the membrane.

    12.21 Relationship Between CNS 5-HT Receptor Type and Effector Mechanism

    Table IV
    Class Receptor Type Synaptic Action Signaling Mechanism
    5-HT1A G protein linked
    Decreased excitability (increased K + conductance)
    1) Decreased cAMP
    2) direct K + channel opening by G proteins
    5-HT1B G protein linked Autoreceptor-mediated decreased 5-HT release Decreased cAMP
    5-HT1E 5-HT1F G protein linked ? Decreased cAMP
    5-HT1D G protein linked Autoreceptor-mediated decreased 5-HT release Decreased cAMP
    5-HT2 G protein linked Increased excitability
    (decreased K + conductance)
    IP3 production
    5-HT4 G protein linked Increased excitability
    (decreased K + conductance)
    Increased cAMP followed by phosphorylation of K + channels
    5-HT3 Ligand gated pentameric cation channel Ligand gated pentameric cation channel Rapid depolarization Increased Na + , K + and Ca 2+ conductance

    Three subtypes of histamine receptors have been identified. All three are G protein linked and all three are present in the CNS as well as the periphery. Thus far, only peripheral H receptors have been characterized (See Table V).

    12.23 Relationship Between CNS and Peripheral Histamine Receptor Type, Location and Effector Mechanism

    H1 receptors mediate the well-known physiological responses to histamine that occur in response to histamine liberation from mast cells. A large number of prescription and over the counter drugs, antihistamines, act by blocking H1 receptors. Because most H1 blockers also have a sedative effect and cause drowsiness, it appears likely that H1 receptors are also present in the CNS. Recently developed H1 blockers that do not cross that blood brain barrier have circumvented the sedative problem.

    The mechanism of action of H1 receptors is the activation of PLC β

    H2 receptors are responsible for the peripheral actions of histamine that are not blocked by H1 antagonists. These receptors are coupled to stimulation of cAMP and are responsible for histamine's stimulation of gastric acid secretion. Recently developed specific H2 receptor blockers, Tagamet and Zantac, are effective clinically for excess secretion of gastric acid. Because these drugs do not cross the blood brain barrier, they have no effects on the CNS.

    H3 histamine receptors are found on histamine nerve terminals where they regulate the release of histamine. There is evidence for these receptors on the terminals of other neurotransmitter types as well, indicating that histamine may regulate the synthesis and secretion of other neurotransmitters. When presynaptic receptors are located on cells other than their own neurotransmitter type they are called heteroreceptors.

    1. Peripherally - contraction of smooth muscle, fluid secretion from respiratory passage cells, increased release of catecholamines from adrenal medulla
    2. CNS, wide spread, especially hypothalamus actions unknown
    1. Peripherally - contraction of smooth muscle and gastric acid secretion
    2. CNS, wide spread, especially the striatum, actions unknown
    1. CNS, wide spread on nerve endings - mediates decreased neurotransmitter release

    12.24 Inactivation of MA Neurotransmitters by Reuptake and Metabolism

    The major mechanism for the inactivation of secreted MA is the reuptake into the nerve terminal from which the MA was released. Under conditions of very high neuronal activity, the MA will also be taken up by neighboring glial cells and will overflow into the capillaries perfusing the CNS. Under all three situations, a portion of the MA will be metabolized by enzymes that inactivate the MA, converting them to inactive products. As described below, measurement of these metabolites is used clinically and in research to monitor the activity of the MA systems.

    12.25 Reuptake of MA Neurotransmitters

    High affinity transport (reuptake) into axon terminals is the main process of inactivation of released monoamines. Reuptake requires sodium ions and a source of energy (e.g., ATP) and is mediated by a protein carrier located on the plasma membrane of the monamine neurons. Tricyclic antidepressants and cocaine inhibit the transporters for DA, NE and 5-HT. Within the past ten years the structure of several MA transporters has been determined and shown to consist of a twelve transmembrane protein with both the N and C terminal ends residing within the cytoplasm (Figure 12.11). The powerful addictive drugs cocaine and amphetamine increase the level of MA neurotransmitters in the extracellular space. Cocaine acts by blocking the transport of MA (Figure 12.11) neurotransmitters into the terminal and as a consequence increases MA in the extracellular space. In contrast, amphetamine reverses the transport direction (Figure 12.11), transporting MA neurotransmitters out of the nerve terminal.

    Figure 12.11
    Reuptake of MA neurotransmitters by a transporter with a twelve transmembrane structure.

    A low affinity uptake of monoamines into surrounding glial cells also inactivates released monoamines. Because this process acts only at very high concentrations of monoamines, it is believed to only come into play when the concentration of released neurotransmitter is very high.

    A portion of released catecholamines diffuse to the extracellular space where monoamine oxidase (MAO) and/or catechol-0-methyl-transferase (COMT) eventually catabolize it. This route of inactivation is more prominent following extremely high levels of catecholaminergic neuronal activity.

    12.26 Metabolism of MA Neurotransmitters

    Catecholamines and 5-HT : The enzymatic metabolism of MA neurotransmitters is carried out by MAO, COMT and histamine methyl transferase. These enzymes are widely distributed in tissues.

    Monoamine Oxidase (MAO) : This metabolic enzyme is located on the outer membrane of the mitochondrion and metabolizes DA, NE and 5-HT by oxidative deamination of (see Figure 12.12) to the corresponding aldehyde (DHPA, DHPGA and 5HIAA, respectively). DHPA and PHPGA are aldehyde intermediates that must be further metabolized by aldehyde reductase or dehydrogenase to alcohols and acids, respectively. These metabolites are excreted (see Table VI below), or further metabolized by methylation through the action of catechol-O-methyltransferase and then excreted (see below). Pargyline, an irreversible inhibitor of MAO, blocks monoamine degradation.


    2. Effects that result from changes in the regulatory domain

    2.1 Phosphorylation and feedback inhibition by catecholamines

    TyrH is different from the other family members in that it has four serine residues in its R domain that become phosphorylated by protein kinases in vivo and in vitro rather than only one. A short summary follows: rat TyrH (rTyrH) is phosphorylated in vivo at ser8, -19, -31, and -40, in response to various nervous stimuli or signaling molecules (position 8 contains a threonine in some species, including human TyrH). The kinases include PKA, PKC, CaMKII, MAPKAP-K2, ERK1, ERK2, MSK1, and PRAK, among others ( Figure 6 ). The in vivo results of those phosphorylation reactions have been well documented (13, 14) and include activation of TyrH. In vivo and in crude lysate samples, rTyrH activity is activated perhaps 20-fold by phosphorylation by PKA and 1.5 to 3-fold by phosphorylation via CaMKII or ERK, respectively (13, 14). In contrast, in vitro studies to determine the mechanism of activation for each serine phosphorylation have recorded less activation: 1) No mechanism of activation has been identified for phosphorylation at position 8 (13, 19). 2) PRAK phosphorylates ser19 alone, and CaMKII phosphorylates ser19 and ser40 with a strong preference for ser19 (13, 14). No effect on Vmax values, KM values, or Kd values for the neurotransmitters has been recorded for ser19 phosphorylation. However, rTyrHpser19pser40 (TyrH phosphorylated at both ser19 and ser40) is activated 1.5 to 2-fold if the enzyme is assayed in the presence of the 14-3-3 proteins (discussed in section 3.1) (13, 14). 3) Ser31 is phosphorylated by ERK1 and 2 and Cdk5, causing a two-fold lowered KM value for tetrahydrobiopterin (13, 14). 4) Ser40 is phosphorylated mainly by PKA with the result of a 2-fold decrease in KM value for tetrahydrobiopterin, a slight increase in Vmax value, and a 300-fold decrease in binding affinity for the catecholamine neurotransmitters, as judged by Kd values obtained from on rates vs off rates of the catecholamines (13, 14). Ser40 is also phosphorylated by MAPKAPK-2, which also labels some ser19 but to a lesser extent (13, 14). Reversal of activation of TyrH occurs when the phosphatase PP2A (and PP2C to a lesser extent) removes the phosphates from ser19, -31, and -40. (25, 26). The major questions concerning TyrH and its multiple phosphorylatable serine residues are considered in turn in the following paragraphs.

    Simplified map of the reactivity of some protein kinases with the serine residues of the R domain of TyrH. Ser40 is modified by PKA, CaMKII, and MAPKAPK-2. Ser31 is modified by ERK1 & 2 and Cdk5. PRAK labels ser19, as do CaMKII, and MAPKAPK-2. Ser8 is modified by ERK1 but it is not certain that the reaction has an effect on TyrH activity. Extensive coverage of these phosphorylation events have been covered in previous reviews (14, 25)

    Does multiple phosphorylation result in additional activation or release from feedback inhibition?

    Why TyrH is phosphorylated in multiple positions is a question that has engaged a number of laboratories. One assumes that a protein would not acquire 3 to 4 different serine residues as modification sites for different kinases, responding to different stimuli, using ATP in every phosphorylation event, without some advantageous molecular effect. Furthermore, since phosphorylation of ser40 by PKA results in at least 20-fold activation, it seems doubtful that 1.5-fold or 2-fold activation after phosphorylation at the other serines tells the whole story of their mechanism, unless it is to fine-tune the effects that are coarsely produced by phosphorylation of ser40 (27, 28).

    A common approach when seeking the molecular mechanism for regulation of a protein by phosphorylation is to employ phosphomimetic variants, that is, variants with serine mutated to glutamate or aspartate. Since phosphorylated TyrH is difficult to obtain in fully and discretely (at one site only) labeled forms, glutamate and aspartate substitution variants have been used, including rTyrHser8glu, -ser19glu, -ser31glu, and -ser19glu/ser40glu (29). While some glutamate or aspartate-substituted enzyme variants do not mimic phosphorylation, rTyrHser40glu is a good mimic of rTyrHpser40 (30, 31), displaying resistance to inhibition by DA to the same extent as rTyrHpser40 (20), and rTyrHser31glu is a good mimic of rTyrHpser31 (29). The double variant rTyrHser19glu/ser40glu was studied because CaMKII and MAPKAPK-2 label both ser19 and -40, so perhaps both must be phosphorylated for full effect from those kinases (32, 33). However, rTyrHser19glu/ser40glu showed the same slight decrease in KM value for tetrahydrobiopterin and slight increase in Vmax compared to the values for wild-type rTyrH as expected for rTyrHpser40 (31) and did not display any additional alterations that would explain why phosphorylation of both serine residues is different than phosphorylating just one. None of the other ser-to-glu variants had altered Michaelis constants compared to wild-type TyrH, nor did any of them show decreased affinity for catecholamines (29), suggesting that phosphorylation at ser19 and ser31 have a different activation mechanism than phosphorylation at ser40.

    Does multiple phosphorylation result in stabilization of the most activated phosphorylated form of the enzyme?

    To determine whether phosphorylation stabilizes TyrH, the stability of the phosphomimetic variants has been tested. Rat TyrHser19glu and –ser31glu were more stable to denaturation at elevated temperature but the effect was not dramatic, a 1.6 to 1.7-fold slower inactivation rate. Rat TyrHser40glu was less stable, denaturing 1.6 times faster than wild-type (29). This suggests that phosphorylation at ser19 and ser31 may serve to stabilize TyrHpser40. Rat TyrHpser40 was less stable than unphosphorylated rTyrH when studied by circular dichroism (34), but IR conformational analysis found rTyrHpser40 to be more stable than unphosphorylated rTyrH (35), so there is some controversy about whether TyrHpser40 is more or less stable than unphosphorylated TyrH.

    Does multiple phosphorylation result in alteration of substrate specificity for other kinases?

    Since phosphorylation attaches a negative charge to a position that had been merely polar (i.e., phosphoserine vs. serine), another possibility for multiple/prior phosphorylation is that once-phosphorylated TyrH might be a better or worse substrate for other kinases. For example, phosphorylation at ser31 by ERK or at ser19 by CaMKII might alter the KM value for TyrH as a substrate for PKA. Relative Vmax/KM (V/K) values are indicators of relative substrate specificity (36), so if a phosphomimetic variant at position 19 or 31 has an altered V/K value as a substrate for PKA, that would indicate a change in substrate specificity for TyrHpser19 or –pser31. However, when the V/K values for the glutamate substitution variants rTyrHser8glu, -ser19glu, and -ser31glu, were measured, none was a better or worse substrate for PKA (37). For PKA the V/K values for wild type rTyrH, rTyrHser19glu and rTyrHser31glu were all around 120 min 𢄡 μM 𢄡 . Therefore phosphorylation at ser19 or ser31 does not affect PKA’s activity at ser40. The results were similar for ERK2 in this experiment the possible impact of prior phosphorylation at ser19 and ser40 on substrate specificity of TyrH for ERK2 was considered, since ERK2 phosphorylates ser31. The V/K values for rTyrH, rTyrHser19glu, rTyrHser40glu, and the double variant rTyrHser19glu/ser40glu, respectively, were all between 0.5 and 0.6 min 𢄡 μM 𢄡 . Apparently prior phosphorylation by PKA does not alter ERK2 reactivity with TyrH.

    Does a first phosphorylation step by a single kinase result in faster reaction at a second serine?

    The previous experiments showed that the kinetic parameters of PKA, CaMKII, MAPKAPK-2, and ERK were not altered by phosphorylation at a different serine by a different kinase. In contrast, using mass spectrometry to determine the rates of phosphorylation of wild-type rTyrH, rTyrHser40ala, and rTyrHpser40 by CaMKII, Bevilaqua et al. calculated that ser40 is more available for phosphorylation if ser19 is already phosphorylated that is, the rate of ser40 phosphorylation by CaMKII is 2-to-3-fold higher if ser19 is already phosphorylated (28). Presumably the resultant rTyrHpser19pser40 is then released from feedback inhibition by catecholamines rTyrHser19glu/ser40glu has the same lowered affinity for DOPA as does rTyrHser40glu (29). At the time of this writing, this result is the most convincing example for hierarchical phosphorylation of TyrH. However, using an ATP to phosphorylate ser19 in order to speed up phosphorylation at ser40 when another kinase exists that only phosphorylates ser40 (PKA) seems inefficient. One possibility is that ser19 phosphorylation occurs to proactively stabilize TyrHpser40. The answer to this question may lie in an added feature of ser19 phosphorylation, that is, the subsequent binding of the 14-3-3 proteins. This feature will be discussed in the section on protein complexes.

    Does phosphorylation result in demonstrable physical changes?

    Bevilaqua et al. (28) have demonstrated rTyrH R domain structural changes upon phosphorylation using gel filtration chromatography they also demonstrated that phosphorylation of ser19 causes an alteration of structure similar to that caused by phosphorylation of ser40. The authors suggested a hinged-movement structure for the R domain of TyrH, similar to the one in Figure 5 , in which the movement is greater when caused by pser40 than by pser19 because ser40 is closer to the pivot point (28).

    Proteolysis has also been used to probe the structure of the R domain of TyrH and changes upon phosphorylation. It has long been known that limited proteolysis of the AAAHs separates the R and C domains (38, 39). Rat TyrH missing 157 and rat PheH missing 141 amino terminal residues retain full activity. More limited proteolysis of TyrH has provided information concerning changes upon phosphorylation. McCulloch (40) showed that rTyrH is cut at 4 positions, namely, arg33, arg37, arg38, and arg49, by trypsin. Rat TyrHpser40 is cut by trypsin faster than non-modified TyrH, meaning that the R domain undergoes a conformational change upon ser40 phosphorylation to a more open position more accessible by the protease. Proteolysis by the Staphylococcus V8 protease, which cuts at positions 27 and 50, was used to probe for alterations in structure due to introduction of negative charges at positions 19, 31, and 40 (29). The glutamate variants rTyrHser19glu, -ser31glu and –ser40glu were used as mimics of phosphorylated TyrH. Only rTyrHser31glu was digested at a different rate than wild-type enzyme (digest was slower), demonstrating that there is a conformational change upon the introduction of a negative charge at position 31. That series of experiments also showed that rTyrH and rTyrHser31glu were digested more quickly by the V8 protease if tyrosine was present. More information about changes associated with tyrosine binding is found below in the section on conformational changes to the catalytic domain.

    Summary Section 2.1: Phosphorylation and feedback inhibition by catecholamines

    TyrH is activated after phosphorylation of any of three serine residues in its regulatory domain. Ser40 is phosphorylated mainly by PKA, resulting in a decrease in affinity for catecholamines. Ser31 is phosphorylated by several kinases, resulting in a decrease in KM value for tetrahydrobiopterin. Ser19 is phosphorylated by enzymes that modify only ser19 or both ser19 and -40, and does not result in activation in the absence of other factors, which will be discussed later under protein interactions. Phosphorylation of ser19 by CaMKII accelerates phosphorylation of ser40 by the same kinase. Any other result of multisite phosphorylation has not yet been established, although stabilization and tighter binding to chaperone proteins are possibilities.

    2.2 Binding by catecholamines

    Dopamine, norepinephrine, and epinephrine are all feedback inhibitors of TyrH, and the biggest alteration of TyrH activity upon ser40 phosphorylation is the change in Kd value for catecholamines. DA affinity for TyrH is 300-fold decreased when the enzyme is phosphorylated (41). Questions concerning feedback inhibition by dopamine, epinephrine, and norepinephrine are considered in turn in the following paragraphs.

    Does catecholamine binding result in demonstrable physical changes?

    The limited proteolysis experiments described above also provided insights into alterations of structure that occur upon dopamine binding (40). McCulloch showed that when dopamine was bound to TyrH, cleavage was much slower than if the enzyme was catecholamine-free. This demonstrates that there is another conformational change in the R domain upon dopamine binding making the peptide less available to trypsin (40). As previously mentioned, proteolysis of TyrHser31glu by the S. aureus V8 protease was slower than proteolysis of wild-type enzyme, but in the presence of dopamine the digest was even slower, demonstrating that the conformational alteration caused by dopamine is separate from the conformational change caused by a negative charge at ser31 (29).

    What are the molecular interactions between TyrH and the catecholamines?

    All three catecholamines bind to the active site via the iron atom, but they only bind if the iron is oxidized therefore binding of catecholamine inhibitors is not a simple equilibrium (30). One might have assumed that the catecholamines bind at the amino acid site, since they are similar in structure to phenylalanine or tyrosine. The proteolysis experiments clearly show that dopamine binds differently than tyrosine. When catecholamines are included in steady-state kinetic assays of TyrH they display competitive binding with respect to tetrahydrobiopterin (42). Several reports propose two binding sites for dopamine with the lower affinity site allowing competitive inhibition with tetrahydrobiopterin (43, 44). Crystal structures show the ligands dopamine, norepinephrine, and epinephrine bound to PheH (there is no crystal structure of TyrH with catecholamines bound) (45). In those structures the catechol moiety is bound in bidentate fashion to the active site iron. Figure 7 shows the overlaid structures of PheH with dopamine (45), PheH with thienylalanine and tetrahydrobiopterin (46), and PheH with its R domain intact (18). (There is no crystal structure of PheH with phenylalanine bound to use for comparison, but thienylalanine is a good analog.) It is clear that in the crystal-packed enzyme, the catecholamine site slightly overlaps the pterin site, not the amino acid site, and the amino end of the catecholamine is close to the R domain, easily close enough for a salt bridge. The amino acid substrate is bound to a separate site from these two sites the analog is bound presumably where phe would bind in the active site pocket, and where tyr would bind in the active site of TyrH. This arrangement would explain how tyrosine and dopamine could both bind in the active site but have different effects on the proteolytic pattern of the R domain. It appears that the R domain has at least three conformations it may be held tightly closed against the C domain when catecholamines are bound, have another form when tyrosine is bound, and a third when it is phosphorylated. A model representing the variability of structure in the R domain is drawn in Figure 8 . The difficulty of crystallizing the R domain suggests that there are others, perhaps many others, when neither substrate nor inhibitor is bound.

    On left, PheH with tetrahydrobiopterin, dopamine, and thienylalanine bound. Three structures were overlaid to create these images: 1PHZ, which contains the R domain 4PAH, which contains catecholamine and 1KW0, which contains tetrahydrobiopterin, and thienylalanine, a phenylalanine analog. The pterin is in blue, the dopamine is in orange, and the thienylalanine is in green. Clearly the dopamine and biopterin sites overlap, and the aromatic amino acid site is separate.

    At right are the same structures in a higher magnification, turned slightly to illustrate the proximity of the catecholamine to the R domain.

    Drawing of three different possible regulatory domain configurations. The regulatory domain is lavender with a mobile yellow loop, and the catalytic domain is light blue. Dopamine is represented by a blue circle. Left, unphosphorylated TyrH the R domain is very flexible. Lower right, TyrH with dopamine bound, chelated to active site iron. The R domain is in a more rigid conformation, less accessible to proteases, and obstructing entrance of substrates. Upper right, TyrHpser40 with salt bridge between some acidic residue and phosphoserine40, exposing the entrance to the active site. TyrHpser40 has released the catecholamine which was bound prior to phosphorylation, the flexible loop is in a different configuration that makes arg37 and arg38 less accessible to trypsin but exposing arg33 and arg49.

    Is catechol binding to the iron in the TyrH active site the only interaction between the two molecules?

    Catecholamines are bound to the iron in the active site of PheH crystals, but there is reason to believe that the other end of the molecule also interacts with the R domain of TyrH. Deletion mutants and site-directed mutants have been studied in the attempt to pinpoint the R domain residue(s) that interact with dopamine. Nakashima et al. (47) have focused on the arginine residues at position 37 and 38. They studied site-directed mutants containing single or double mutations of these arginine residues to glycine or glutamate. When both were mutated TyrH activity was not decreased as much by dopamine, judging by activities of the TyrH variants preincubated with 1, 10, and 100 μM dopamine, but Ki values were not measured. This is consistent with the catechol end of dopamine binding to the iron, but the amino end not being able to form an association with the R domain when arg37 and arg38 are missing. Piper and Daubner (48) made deletion variants of rTyrH lacking the first 32 (TyrH𹐲), the first 68 (TyrH𹑨), the first 76, or the first 120 amino acids. The deletion variants were tested for inhibition by preincubation with stoichiometric amounts of dopamine TyrH𹐲 was 90% inhibited by dopamine, but TyrH𹑨 and the other truncates were not inhibited (unpublished observations). Furthermore, when dopamine binding and release rates were investigated using the method of Ramsey and Fitzpatrick (41), dopamine was not released from TyrH𹐲 but was rapidly released from TyrH𹑨. That TyrH𹐲 interacts with dopamine but TyrH𹑨 does not suggests that some amino acid residue or some three-dimensional structure between positions 32 and 68 is important for dopamine binding. This is consistent with the Nakashima result that arg37 and arg38 are involved (47). However, it is not clear how arginine residues would link the dopamine amino group to the R domain, since all would presumably be positively charged at pH 7. A thorough study of the Kd values of an array of catechols to TyrH suggested that the amino group of dopamine is imperative for tight binding and the carboxyl group is not needed. Dopamine binds 1000-fold more tightly than DOPA, and dihydroxyphenylacetate binds 100-fold times less tightly than DOPA (30). Perhaps arg37 and arg38 are important for the tertiary structure of the R domain. Supporting this idea, the molecular mechanics and dynamics work of Alieva et al. (49) proposed that arg37 is necessary for an important β-turn in the R domain. A carboxylate residue could bind the catecholamine amino group, but alanine and glutamine substitution for the negatively-charged residues between position 32 and 68 (TyrHglu43, -asp44, -glu48, and glu-50) did not identify any carboxylate in the region important for dopamine binding. These TyrH variants were all inhibited by preincubation with stoichiometric amounts of dopamine but Kd or Ki values were not measured (Daubner, unpublished observations).

    Recently TyrH has been studied using hydrogen-deuterium exchange followed by proteolysis and mass spectrometry (50). The technique identifies portions of proteins that readily exchange hydrogens from the peptide backbone with deuterium in solvent, and in this way, identifies regions of proteins that are more flexible (51). This technique established that 2 peptides encompassing residues 35� were less likely to incorporate deuterium if TyrH was preincubated with dopamine, that is, these peptides are more shielded from solvent when dopamine is bound. The finding corroborates the proteolysis experiments that showed that dopamine-bound R domain was less available to trypsin or V8 protease. It also corroborates the Nakashima conclusion, since arg37 and arg38 lie within the peptide whose structure becomes less exposed in the presence of dopamine. The peptides containing residues 35� were not detected when the experiment was done with TyrHpser40, so a comparison of this peptide to its phosphorylated form is not possible. A peptide in the catalytic domain showed changes after phosphorylation. The peptide containing residues 295� (leu29-ser296-ala297-arg298-asp299) became more exposed to deuterated solvent upon treatment with PKA, presumably because the movement of the R domain away from the opening to the active site exposed that peptide. This peptide’s position with respect to the R domain in PheH is shown in Figure 9 .

    Peptides of PheH corresponding to the peptides of TyrH identified by hydrogen-deuterium exchange. The ribbon is derived from crystals of PheH missing 19 amino terminal amino acids (1PHZ). Overlaid on that structure are the structures of PheH with dopamine (5PAH), BH4 and thienylalanine (1KW0), with only the small molecules shown. Thienylalanine is in green, dopamine in magenta with its amino nitrogen in blue, and tetrahydrobiopterin is blue. The region of the R domain that is homologous to TyrH’s gly36-glu50 is colored yellow. The region of PheH that is homologous to TyrH positions 295� is colored red.

    Summary Section 2.2: Binding of catecholamines

    TyrH phosphorylation at ser40 results in a lowered affinity for catecholamines. Dopamine binds to TyrH in the active site in a location that overlaps tetrahydrobiopterin dopamine binding to TyrH results in an altered conformation for the R domain that prohibits entry of substrates into the active site. Arg37 and arg38 are necessary for inhibition by dopamine, perhaps by determining the overall three-dimensional structure of the R domain. Inhibition by the catechols is dependent on the presence of an amino group, not a carboxylate group.

    2.3 R domain differences between rat TyrH and human TyrH

    Human TyrH is more complex than the rat enzyme. Humans have 4 isoforms of TyrH that differ in primary sequence just prior to ser31, the result of different splicing of the pre-mRNA. Referred to as hTH1, hTH2, etc, they differ in the length of the R domain. hTH1 is the same size as rat TyrH and is very similar in amino acid sequence. hTH2 contains 4 additional amino acids after met30 (VRGQ), hTH3 has 27 additional amino acids (GAPGPSLTGSPWPGTAAPAASYTPTPR), and hTH4 has all 31 additional amino acids (VRGQGAPGPSLTGSPWPGTAAPAASYTPTPR) (52, 53). A drawing of the differences in the R domain of the human enzymes is shown in Figure 10 . The insertion of new amino acids between met30 and ser31 alters the protein just prior to the serine that is phosphorylated by ERK1 and 2, and also increases the spacing between ser19 and ser40. It also alters the numbering of ser31 and ser40, but the homologs of ser31 and ser40 in isoforms 2𠄴 will be refered to as ser31 and ser40 for the sake of clarity. No complete model has yet emerged to explain the physiological advantage for the existence of the four human isoforms. They have comparable Vmax and KM values. They are all inhibited by dopamine, binding 2 to 3-fold tighter than the rat enzymes, and all bind DOPA approximately 6-fold more tightly than the rat enzyme (54). Because of the loss of the ERK consensus sequence in hTH2, its homolog to ser31 is not phosphorylated by ERK (55), but the repercussions of that loss are not known.

    Drawing of the R domains of the human isoforms of TyrH illustrating their structural differences. Amino termini are to the left. hTH1 is identical in length to rat TyrH, and appears first, with the regulatory serines shown. hTH2 has four additional amino acids after met30, and those amino acids are shown as a short pink segment of the R domain. hTH3 has 27 additional amino acids, shown as a yellow segment, included after met30. hTH4 has both additional segments so has 31 amino acids more than hTH1. These additional amino acids are included via alternative mRNA splicing. Since the additional amino acids come immediately before ser31, the serine residues homologous to ser31 and ser40 have different numbers in hTH’s 2, 3, and 4, but are rarely referred to by these numbers.

    Summary Section 2.3: R domain differences between rat and human TyrH

    Human TyrH comes in four isoforms isoform 1 is very similar to rat TyrH. Isoforms 2𠄴 contain additional amino acids in the very region of the R domain where the regulatory serines are positioned. The human isoforms have slightly different affinities for DOPA and dopamine than rat TyrH, and slightly different affinities from each other as well. To be discussed in section 2, there may be some differences in binding affinity for other proteins.


    Endogenous Catecholamines in Immune Cells: Discovery, Functions And Clinical Potential as Therapeutic Targets

    The catecholamines dopamine, noradrenaline and adrenaline are well established neurotransmitters and neurohormones. Sympathoadrenergic pathways are pivotal to the communication between the nervous system and the immune system, and the role of dopaminergic pathways in the modulation of immune response is being increasingly unveiled. However, over the last two decades, evidence has accumulated regarding the ability of immune cells themselves to produce and utilize dopamine, noradrenaline and adrenaline. Endogenous catecholamines in immune cells represent a novel and emerging area of research with wide implications. As many dopaminergic and adrenergic agents are already in clinical use for several non-immune indications and with a usually favorable tolerability profile, this may represent an extremely attractive source of immunomodulating agents with significant therapeutic potential.

    1. Physiology and pharmacology of catecholamines: an overview

    Catecholamines are a family of chemical compounds containing a catechol or 3,4-dihydroxyphenyl group and an amine function. Dopamine, noradrenaline and adrenaline are the most abundant and important catecholamines in the human body and are all produced from l-tyrosine, a non-essential amino acid which is either obtained from dietary proteins or synthesized from the essential amino acid phenylalanine by the enzyme phenylalanine hydroxylase. l-tyrosine is converted to l-3,4-dihydroxyphenylalanine (l-DOPA) by tyrosine hydroxylase (TH, also known as tyrosine 3-monooxygenase, EC 1.14.16.2), which is the rate-limiting enzyme in the synthesis of catecholamines. The enzyme aromatic l-amino acid decarboxylase (EC 4.1.1.28, also known as DOPA decarboxylase) catalyzes the conversion of l-DOPA to dopamine. Noradrenaline is in turn synthesized from dopamine by dopamine β-hydroxylase (DBH, EC 1.14.17.1) and is converted to adrenaline by phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) (see Biosynthesis of dopamine, noradrenaline and adrenaline Wikimedia Commons Catecholamines biosynthesisAuthor: NEUROtiker, last accessed: 2 October 2013).

    It has also been suggested that d-DOPA, the stereoisomer of l-DOPA, may be oxidized by the enzyme d-amino acid oxidase (DAO, EC 1.4.3.3) and then converted to dopamine via an alternative biosynthetic pathway however the relevance of this pathway remains to be established [1].

    Dopamine and noradrenaline (and, to a lesser extent, adrenaline) act as neurotransmitters in the central nervous system (CNS). Noradrenaline is also the main neurotransmitter in postganglionic neurons on the sympathetic nervous systems, while adrenaline (and, to a lesser extent, noradrenaline) is the neurohormone secreted by chromaffin cells in the medulla of adrenal glands.

    Dopamine: Four major dopaminergic pathways exist in the CNS: mesolimbic (associated with reward and learned behaviors), mesocortical (involved in cognitive functions including motivation, reward, emotion, and impulse control), nigrostriatal (affecting movement) and tuberoinfundibular (regulating prolactin secretion from the pituitary).

    In the periphery, dopamine acts as paracrine/autocrine transmitter in the kidney, increasing natriuresis, renal blood flow and glomerular filtration. Circulating dopamine causes vasodilatation and reduced cardiac afterload, resulting in decreased blood pressure and increased cardiac contractility. Higher concentrations of dopamine however may act not only on dopaminergic receptors but also on β- and α-adrenoceptors (ARs): the former contribute to increased cardiac contractility while the latter result in vasoconstriction and increased blood pressure. Detailed information about anatomy, physiology and pharmacology of dopamine in the nervous system can be found in [2].

    Noradrenaline and adrenaline: Noradrenaline and, to a lesser extent, adrenaline act as neurotransmitters in the CNS and in the peripheral nervous system. Chromaffin cells in medulla of adrenal glands also produce adrenaline (

    80% in humans) and noradrenaline (

    20%), which are directly released into the blood. In the CNS, the most important noradrenergic nucleus is the locus coeruleus (LC), from which axons project to the hippocampus, septum, hypothalamus and thalamus, cortex and amygdala, to the cerebellum, and also to the spinal cord. The LC is involved in attention, arousal and vigilance to external stimuli, and it regulates hunger by stimulating feeding behaviors. In the periphery, noradrenaline is the main transmitter of most autonomic sympathetic postganglionic fibers, and its main actions include: smooth muscle contraction in blood vessels supplying skin, kidney, and mucous membranes, stimulation of exocrine glands such as salivary and sweat glands, smooth muscle relaxation in the gut wall, bronchi and blood vessels supplying skeletal muscle, increased heart rate and force of contraction, as well as metabolic (increased glycogenolysis in liver and muscle, lipolysis in adipose tissue) and endocrine actions (modulation of insulin and renin secretion). Noradrenaline and adrenaline neurochemistry, anatomy and physiology are extensively discussed in [2].

    1.1. Dopaminergic receptors

    Receptors for dopamine, noradrenaline and adrenaline belong to the 7-transmembrane, G-protein coupled receptors family. Dopamine in particular acts on 5 different dopaminergic receptors (DRs) grouped into two families according to their pharmacological profile and main second messenger coupling: the D1-like (D1 and D5) which activate adenylate cyclase and the D2-like (D2, D3 and D4) which inhibit adenylate cyclase [3,4] (Table 1).

    DR ligands are currently used in the treatment of several non-immune conditions: agonists are used in the treatment of Parkinson’s disease, restless leg syndrome, and hyperprolactinemia, while antagonists are mainly used as antipsychotics and antiemetics. Physiopharmacology of DRs is summarized in Table 1, together with examples of DR ligands used in therapeutics, while detailed and continuously updated information about DR is provided in [4].

    Noradrenaline and adrenaline act on 9 different ARs, which include three major types – α1, α2 and β – each further divided into three subtypes [5] (Table 2).

    AR agonists and antagonists are used to treat a variety of diseases, including hypertension, angina pectoris, congestive heart failure, asthma, depression, benign prostatic hypertrophy, and glaucoma. Additional conditions where AR ligands proved useful include shock, premature labor and opioid withdrawal, and as adjunct medications in general anesthesia. Physiopharmacology of α- and β-ARs together with examples of ligands used in therapeutics are listed in Table 2 and in Table 3, while detailed and continuously updated information is provided in [5].

    1.3. Modulation of dopaminergic and adrenergic pathways by indirectly acting agents

    Besides classical receptor-targeted pharmacology, dopaminergic and adrenergic pathways offer a number of additional targets which include all the steps involved in catecholamine synthesis, storage and release, uptake and metabolism. Potential targets and examples of drugs already in use for non-immune indications (e.g. cardiovascular, neurologic, neuropsychiatric) are listed in Table 4.

    2. Dopaminergic and adrenergic modulation of immunity

    The immune effects of dopamine as well as of noradrenaline and adrenaline have been the subject of several comprehensive reviews [6-14].

    Adrenergic pathways modulating the immune response have been extensively investigated, since the identification of the sympathoadrenergic system as one of the two major pathways (the hypothalamic-pituitary-adrenal axis being the other one) responsible for the CNS-immune system cross-talk. In this context, sympathoadrenergic terminals release noradrenaline which acts on β2-ARs on immune cells inducing both inhibition of T helper (Th) 1 proinflammatory cytokines (e.g. IFN-γ, IL-12, TNF-α) and enhancement of Th2 cytokines (e.g. IL-10 and TGF-β), finally resulting in antiinflammatory effects [7,8]. Activation of β-ARs on human lymphocytes under specific conditions may however result also in stimulation of the immune response: for instance, noradrenaline may promote IL-12-mediated differentiation of naive CD4+ T cells into Th1 effector cells, and increase the amount of IFN-γ produced by Th1 cells [15]. Inhibitory β2-ARs are also expressed by monocytes/macrophages and neutrophils, as well as in CNS resident immune cells such as microglia and astrocytes. Proinflammatory responses may result also from activation of α1-ARs on human macrophages [16] or α2-ARs on rodent phagocytes [17], however in comparison to β-ARs, α-ARs in immune cells have so far received little consideration. A detailed review of adrenergic mechanisms modulating the immune response, including an extensive summary of α- and β-AR expression and function on cells of the innate and adaptive immune response, has been published recently [13].

    Dopaminergic pathways in the modulation of immunity began only recently to receive specific attention [6,10,11,14]. Evidence exists that DR are expressed possibly in all human immune cells (including T and B cells, dendritic cells, macrophages, microglia, neutrophils and NK cells), and that immune cells can ‘meet’ dopamine in the brain as well as in blood, lymphoid organs and in several other peripheral tissues, such as the kidney and the liver (reviewed in [11]). Dopamine is also emerging as a regulator of dendritic cell and T cell physiology [18]. Based on available evidence, it has been proposed that dopamine may result in preferential activation of resting T cells and in inhibition of stimulated T cells [11]. For instance, in naive human T cells dopamine acts on DR D2/D3 receptors to induce β1 integrin-dependent adhesion to fibronectin [19], and on DR D2/D3 and D1/D5 receptors to increase TNF-α and IL-10 [20]. The effects of dopamine are likely specific for the various subsets of immune cells: for instance, in human CD4+CD25 high T lymphocytes activation of D1-like receptors inhibits their ability to suppress the activity of effector T cells [21].

    3. The discovery of endogenous catecholamines in immune cells

    The first report showing the occurrence of endogenous catecholamines in immune cells dates back to 1994, when Jonas Bergquist and co-workers identified catecholamines and their metabolites in single lymphocytes and extracts of T- and B-cell clones by the use of capillary electrophoresis with electrochemical detection (a technique, at that time emerging, capable of rapidly determining multiple chemical species in pico-femtoliter biological samples). The endogenous origin of catecholamines was supported by pharmacological evidence showing that inhibition of TH reduced catecholamine levels, while the functional relevance of the observation was suggested by the ability of either dopamine or its precursor l-DOPA to inhibit in a concentration-dependent fashion lymphocyte proliferation and differentiation [22].

    Two years before that, Georges J.M. Maestroni , working at the Center for Experimental Pathology in the Istituto Cantonale di Patologia of Locarno (CH), had reported for the first time that the sympathetic nervous system may modulate hematopoietic reconstitution after syngeneic bone marrow transplantation in mice [23]. Maestroni showed that chemical sympathectomy by 6-hydroxydopamine significantly increased the number of peripheral blood leukocytes after syngeneic bone marrow transplantation, and that AR ligands could modulate such effect. We met Georges Maestroni shortly after, in 1993, and our first collaborative paper was published in 1997, reporting the results of catecholamine measurement in the mouse bone marrow by means of HPLC with electrochemical detection, showing that dopamine, noradrenaline and adrenaline could be detected at this level and that noradrenaline was decreased by treatment with 6-hydroxydopamine and increased after pargyline, thus concluding that noradrenaline in the bone marrow originates mainly from sympathetic nerve endings and is metabolized through specific enzymatic pathways, while adrenaline and dopamine may originate from other sources [24]. Noradrenaline and dopamine in murine bone marrow exhibited a daily rhythmicity, with peak values occurring at night. The rhythm was disrupted by chemical sympathectomy, whereas adrenaline showed no rhythmicity or sensitivity to 6-hydroxydopamine. Noradrenaline (but not dopamine or adrenaline) was positively associated with the proportion of cells in the G2/M and S phases of the cell cycle [25]. After chemical sympathectomy catecholamine levels in the bone marrow were reduced but not abolished, an observation which prompted us to investigate their occurrence in both short-term and long-term bone marrow cultures as well as in human or murine B lymphoid cell lines, where all the three catecholamines were identified in significant amounts [25,26]. The obvious conclusion was that endogenous catecholamines in the bone marrow have both neural and cellular origins, therefore we started a systematic investigation of endogenous catecholamines in the immune system, with particular regard to human immune cells, by use of an HPLC method specifically adapted to the purpose [27] (Figure 1).

    Shortly thereafter, we reported the occurrence of endogenous catecholamines in human peripheral blood mononuclear cells (PBMCs) [28] as well as in human granulocytes [29]. It was however a serendipitous observation which soon after led us to understand that TH-dependent catecholamine production in human lymphocytes could be hugely upregulated upon cell stimulation [30]. Increased production of catecholamines in activated lymphocytes led us to suggest a preferential involvement of catecholaminergic pathways in the functional modulation of activated cells and to propose that accumulated catecholamines could represent a supply of mediators to be released upon appropriate stimulation and which in turn may act upon lymphocytes themselves and/or upon neighboring cells [31]. In the following section, evidence so far available regarding the occurrence and the functional role of endogenous dopamine, noradrenaline and adrenaline in immune cells will be summarized and examined, and subsequently the relevance of such intrinsic dopaminergic and adrenergic pathways in the pathogenesis and progression of human disease as well as in the response to drug therapeutics will be discussed.

    4. Presence of catecholamines in immune cells

    4.1. Intracellular content and endogenous synthesis

    Dopamine, noradrenaline and adrenaline (together with their major metabolites) have been identified and measured in several immune cell types, including: murine lymphocytes [32], peritoneal macrophages [33], bone marrow derived mast cells [34], splenic T and B cells [35], human peripheral blood mononuclear cells [28,30,36-38], various lymphocyte subsets [22,27], including CD4+CD25+ regulatory T lymphocytes [21], granulocytes [29], and hematopoietic cell lines [27]. Data concerning endogenous catecholamine levels and production in human immune cells are summarized in Table 5 and Table 6.

    Catecholamines in immune cells are likely synthesized by the classical biosynthetic pathway (Figure 1), as indicated by the expression in both murine and human lymphocytes of the enzyme TH, the first and rate-limiting enzyme in the synthesis of catecholamines [30,35,39,40]. In human PBMCs, TH is strongly upregulated after cell activation, an event which occurs 6-8 h after the addition of phytohemagglutinin (PHA) and which is followed, after 48-72 h, by a sharp increase of intracellular catecholamines, which is prevented by the TH inhibitor a-methyl-p-tyrosine, as well as by the RNA-polymerase inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide [30]. The expression and the activity of the other enzymes involved in the catecholamine biosynthetic pathway have been not yet fully characterized, although circumstantial evidence has been published regarding both DBH [41] and PNMT [42,43]. In rat splenic T and B cells a complex modulation of TH and PNMT expression and activity has been recently shown following stress exposure [35]. We recently identified DBH in human PBMCs by immunocytochemistry, close to the vesicular monoamine transporter (VMAT) type 2 as it could be expected in case the molecular organization of catecholamine storages in immune cells would resemble the neuronal ones (Cosentino M., De Bernardi S. and co-workers, unpublished data) (Figures 1).

    The increase of intracellular catecholamines after mitogen stimulation occurs through protein kinase C activation and the contribution of intracellular Ca ++ -dependent mechanisms [30], and is in line with the reported upregulation of ARs occurring in lymphocytes following mitogen, glucocorticoid or proinflammatory cytokine treatment (see e.g. [44,45]) as well as with the upregulation of DRs after mitogen stimulation (Cosentino M., Kustrimovic N. and co-workers, unpublished data).

    Figure 1. Expression of VMAT2 and DBH in human PBMCs. (Cosentino M., De Bernardi S. and co-workers, unpublished data)

    Altogether, available evidence suggests a preferential involvement of intracellular catecholamine-operated pathways in activated immune cells, although differences are likely to occur among distinct cell subsets. For instance, we reported that in human peripheral blood mononuclear cells activated with PHA, the expression of TH mRNA as well as catecholamine production occurs only in T and B lymphocytes and is reduced by dopamine (but not by noradrenaline or adrenaline) through dopaminergic D1-like receptor-dependent mechanisms which include inhibition of TH gene transcription [46].

    The proinflammatory cytokine IFN-γ exerts similar effects and its action is counteracted by IFN-β [47]. TH expression and catecholamine production are on the contrary enhanced by agents that induce catecholamine release (see below). Nonetheless, CD4 + CD25 high regulatory T lymphocytes, which are specialized T cells playing crucial roles in the control of immune homeostasis and express several surface markers of activation, also constitutively express TH and contain substantial amounts of dopamine, noradrenaline and adrenaline, together with ARs and DRs [21] (see section 5. for a detailed discussion about the functional role of intracellular catecholamines in different types of immune cells).

    4.2. Storage and release

    Agents able to effectively induce the release of catecholamines from human lymphocytes include reserpine [21,27,28], IFN-β [47] and elevation of extracellular K + concentrations ([K + ]e) [31]. Reserpine acts by irreversibly blocking VMAT [48]. In human cells there are two types of VMAT, VMAT1 and VMAT2, which are expressed in neural and neuroendocrine cells [49]. Preliminary evidence indeed suggests their occurrence also in immune cells and tissues such as rat thymus and spleen [50] and possibly also human peripheral blood lymphocytes [51]. Our group recently characterized the expression of VMAT2 in human PBMCs (Cosentino M., De Bernardi S. and co-workers, unpublished data) (Figures 3 and 4). Co-localization of DBH and VMAT2 suggests that intracellular compartments containing catecholamines in immune cells resemble those in synaptic terminals. DBH is specifically expressed in noradrenaline-containing neurons, and it is the only catecholamine-synthesizing enzyme localized within synaptic vesicles, where it occurs in both soluble and membrane-bound forms [52]. Synaptic-like vesicles have never been identified in immune cells, where nonetheless regulated secretion by means of specialized organelles has been extensively characterized in several cell types, including CD8+ T cells, NK cells as well as CD4+ T cells [53]. Interestingly, available information suggests that the molecular mechanisms of granule exocytosis in catecholamine-secreting chromaffin cells in the adrenal medulla and in cytotoxic T lymphocytes share many similar features [54].

    Additional similarities between immune cells and neurons include the upregulation of TH expression and catecholamine production following catecholamine release [27,47], which is similar to the increased activity of neuronal catecholamine-synthesizing pathways following depletion with reserpine (see e.g. [55]).

    4.3. Catecholamine-metabolizing enzymes

    Dopamine, noradrenaline and adrenaline are metabolized through monoamine oxidase (MAO)- and catechol-O-methyl transferase (COMT)-mediated pathways. Occurrence of both MAO and COMT in immune cells is indirectly suggested by the intracellular presence of all the main catecholamine metabolites [21,22,27-29,33,34,36,38] (Figure 2).

    MAO activity in immune cells has been sometimes studied as a marker of neurodegenerative and neuropsychiatric disease [56,57]. MAO activity, predominantly of the B type, occurs in both human granulocytes and lymphocytes [58-60]. Support to its functional relevance has been provided mainly by use of pargyline, an irreversible MAO-B inhibitor, which leads to increased catecholamine levels in concanavalin A (Con A)-stimulated rodent lymphocytes [61], in human PBMCs [28] and in human granulocytes [29]. Recently, it has been reported that MAO-A is expressed in human monocytes in particular after incubation with IL-4, and that upregulation of MAO-A in these cells may contribute in switching naive monocytes into a resolving phenotype [62,63].

    In comparison to MAO, investigations on COMT in immune cells have been so far very limited [64].

    4.4. Membrane transporters

    Catecholamine membrane transporters include DAT (DopAmine Transporter) and NET (NorEpinephrine Transporter), both belonging to the solute carrier 6 (SLC6) gene family [65]. Interestingly, the affinity of noradrenaline and dopamine for NET and for DAT is about the same (see e.g. the PDSP Ki database – http://pdsp.med.unc.edu/pdsp.php), thus allowing sympathoadrenergic nerve terminals (which express NET but not DAT) to take up dopamine from the extracellular environment [66]. Evidence for the expression and the functional relevance of DAT in immune cells has been recently revised [67]. On the contrary, so far the only indirect evidence for the presence of NET in immune cells was provided nearly three decades ago, when Audus and Gordon [68] described a single population of desipramine-binding sites with an apparent dissociation constant (Kd) of about 0.4 nM in murine lymphocytes. Incubation of human PBMCs with the NET inhibitor desipramine or with the DAT inhibitor GBR 12909 results in increased extracellular levels of both dopamine and noradrenaline [28], an observation which is compatible with the occurrence of both transporters on the human lymphocyte membrane. Extensive evidence exists regarding the immunomodulating effects of monoamine uptake inhibitors (see e.g. [69]), however it remains to be established whether such effects may be attributed at least in part to a direct action on DAT and/or NET expressed on immune cells.

    5. Functional role of immune cells-derived catecholamines

    Experimental strategies to investigate the role of endogenous catecholamines in immune cells include: (i) interference with synthesis/degradation (ii) interference with intracellular storage/release/uptake (iii) effect of receptor blockade. Modulation of endogenous catecholamines can be obtained also by use of non-pharmacological approaches, e.g., suppression of expression of key proteins – TH, VMAT, etc. – by means of gene silencing techniques.

    Several lines of evidence suggest that endogenous noradrenaline and adrenaline subserve autocrine/paracrine regulatory loops in mouse peritoneal macrophages. Spengler et al. [33] showed that in these cells LPS-induced production of TNF-α was increased by the β-AR selective antagonist propranolol and decreased by the a2-AR selective antagonist idazoxan. In the same study intracellular noradrenaline was identified in mouse macrophages. Results were later confirmed by Chou et al. [70], who showed that the effect on TNF-α was even more pronounced in macrophages obtained from rats with streptococcal-cell-wall-induced arthritis. Evidence for endogenous catecholamines acting on α2-ARs was also reported in rodent phagocytes, where exposure to LPS resulted in catecholamine release together with induction of catecholamine-generating and degrading enzymes [17]. Blockade of α2-ARs or pharmacological inhibition of catecholamine synthesis suppressed lung inflammation in two rodent models of acute lung injury while α2-AR agonist or inhibition of catecholamine-degrading enzymes induced increased inflammation [17] (see also section 5.3). Phagocytes from adrenalectomized rodents showed greatly enhanced catecholamine release together with increased expression of TH and DBH, and in these cells noradrenaline and adrenaline via α2-ARs seemed to directly activate NFκB, thus enhancing the release of TNF-α, IL-1β and IL-6 and the overall acute inflammatory response [71].

    Recently, Gaskill et al. [72] showed that primary human monocyte-derived macrophages express mRNA for all the five subtypes of DRs, and that DR D3 and DR D4 are expressed on the plasma membrane. Monocyte-derived macrophages also express mRNA for DAT, VMAT2, TH and DOPA decarboxylase. DAT was shown to be expressed on the plasma membrane, VMAT2 on cellular membranes and TH and DOPA decarboxylase were in the cytosol. In the same study it was also shown that exogenous dopamine increased IL-6 and CCL2 production in unstimulated macrophages, and it increased IL-6, CCL2, CXCL8 and IL-10 and decreased TNF-α in LPS-stimulated macrophages [72]. Whether the same effect can be exerted by endogenous dopamine produced by macrophages however could only be indirectly inferred by published results.

    Endogenous catecholamines play a functional role also in cells of the adaptive immunity. In particular, in rodent lymphocytes Qiu et al. [73] reported that stimulation with Con A markedly increased both TH expression and catecholamine content, and that pharmacological inhibition of TH significantly enhanced Con A-induced IL-2 production. They concluded that endogenous catecholamines exert a tonic inhibition on the production of IL-2. Qiu et al. [61] subsequently showed that pharmacological inhibition of TH increased proliferation of rodent lymphocytes, which on the contrary was decreased by the MAO inhibitor pargyline. Pargyline also increased intracellular cAMP, the second messenger acted upon by β-ARs, and β-AR antagonists prevented the effect of pargyline, suggesting that the effect depended upon increased levels of catecholamines (likely, noradrenaline and/or adrenaline) in turn acting on β-ARs. Subsequently, the same group [74] isolated lymphocytes from the mesenteric lymph nodes of mice and stimulated the cells with Con A, showing that inhibition of TH with a-methyl-p-tyrosine reduced catecholamines both in lymphocytes and in supernatants, and upregulated expression of mRNAs and proteins of T-box expressed in T cells (T-bet) and IFN-γ but downregulated expression of mRNAs and proteins of GATA binding protein 3 (GATA-3) and IL-4. In contrast, pargyline increased intracellular and supernatant catecholamines and downregulated expression of T-bet and IFN-γ but upregulated expression of GATA-3 and IL-4. Similar results were obtained in CD4+ T lymphocytes isolated from the mesenteric lymph nodes of mice and transfected with recombinant TH miRNA expression vector (pcDNA6.2-GW/EmGFPmiR-TH) to inhibit the expression of TH [75], thus suggesting that catecholamines synthesized and secreted by lymphocytes regulate differentiation and function of Th cells, with an effect facilitating the shift of Th1/Th2 balance toward Th2 polarization.

    As regards to human lymphocytes, Knudsen et al. [76] initially showed that intracellular levels of noradrenaline and adrenaline in circulating lymphocytes from healthy subjects strongly correlated with both basal and isoprenaline-stimulated intracellular cAMP. Variability in endogenous lymphocyte concentration of adrenaline also correlated with concomitant changes in the number of NK (CD3-CD56+) cells and cAMP in a subgroup of subjects [76]. Our group a few years later showed that in human stimulated PBMCs inhibition of catecholamine synthesis with a-methyl-p-tyrosine resulted in decreased activation-induced apoptosis [39]. Similar findings were subsequently obtained in rodent lymphocytes activated with Con A, where the proportion of apoptotic cells as well as the expression of apoptosis-related genes and proteins, Bax, Fas, Fas-Ligand and caspase-3 were decreased by a-methyl-p-tyrosine but increased by the MAO inhibitor pargyline, which on the contrary decreased the expression of the antiapoptotic protein Bcl-2 [77]. This effect was mediated by cAMP-PKA- and PLC-PKC-linked CREB-Smac/DIABLO pathways coupled to a1– and b2-ARs [78]. Among human lymphocytes, CD4+CD25 high regulatory T cells, which are specialized T cells playing a key role in the control of immune homeostasis, have been shown to constitutively express TH and to contain substantial amounts of dopamine, noradrenaline and adrenaline, which are released upon treatment with reserpine. This suggests that catecholamines are stored in the cells by means of VMAT-dependent mechanisms, as indicated also by the presence of mRNA for both VMAT1 and 2 [21]. Functional experiments showed that catecholamine release in these cells results in reduced production of IL-10 and TGF-b, and in down-regulation of CD4+CD25 high T cell-dependent inhibition of effector T lymphocyte proliferation, which occurs without affecting the production of TNF-a or IFN-g. Both CD4+CD25 high T cells and effector T lymphocytes expressed on the cell membrane D1-like and D2-like DRs to a similar extent (12%-29% of the cells) however dopamine increased intracellular cAMP only in CD4+CD25 high T cells. Catecholamine-dependent down-regulation of CD4+CD25 high T cells was selectively reversed by pharmacological blockade of D1-like DRs, thus suggesting that in human CD4+CD25 high T cells endogenous catecholamines subserve an autocrine/paracrine loop involving dopaminergic pathways and resulting in down-regulation of CD4+CD25 high T cell regulatory functions [21].

    5.3. Evidence from in vivo models

    Flierl et al. [17] showed that in rats with acute lung injury stimulation of α2-ARs by either endogenous catecholamines or by exogenous agonists increased lung inflammation, which on the contrary was suppressed by α2-AR antagonists or inhibitors of catecholamine synthesizing enzymes. Findings were subsequently confirmed and extended in a rodent model of immune complex-induced acute lung injury, where it was also shown that the α2-AR-mediated increase of the severity of acute lung injury was enhanced by adrenalectomy [71]. Based on experimental evidence, it was suggested that the observed effect were dependent upon the catecholamines produced by phagocytic cells (neutrophils and macrophages) and in particular that both adrenaline and noradrenaline directly activate NFkB in macrophages, causing enhanced release of proinflammatory cytokines like TNF-α, IL-1β and IL-6.

    In humans so far at least indirect evidence exists about the in vivo role of intracellular catecholamines in immune cells, in particular in multiple sclerosis (MS).

    Peripheral blood lymphocyte levels of adrenaline seem to be higher in the first attack of MS whilst noradrenaline levels may be lower during remissions [79]. In stimulated lymphocytes from MS patients, no difference was observed in noradrenaline or adrenaline levels in comparison to cells from healthy controls, however cells from patients with chronic-progressive MS or relapsing-remitting MS in relapse produced less dopamine [39]. Endogenous catecholamines play a role in activation-induced apoptosis of lymphocytes [39], and the findings in cells from MS patients may thus be connected to the impairment of apoptotic mechanisms, possibly contributing to the survival of autoreactive cells in MS [80-83].

    It was subsequently reported that in lymphocytes from MS patients treated with IFN-β for 12 months the production of catecholamines greatly increased and was less sensitive to IFN-γ-induced inhibition. Expression of mRNA for TH, β2-ARs and DR D5 was already enhanced after 1 month and further increased up to 6-12 months of treatment [84]. During treatment with IFN-β dopaminergic pathways in circulating lymphocytes likely shift from a prevalent D2-like DR operated modulation in untreated patients towards a prevalent D1-like DR modulation after IFN-β. These changes may be relevant for the therapeutic response in MS since D1-like DR D5 likely mediate the inhibitory effects of dopamine on proliferation and cytotoxycity in human CD4+ and CD8+ T cells [85], whereas activation of either the D2-like DR D2 or D3 may induce T cell proliferation and adhesion [19].

    Interestingly, specific lymphocyte subsets may have different arrangements of dopaminergic pathways: for instance, D1-like DR (likely D5) play a role in the inhibition of human CD4+CD25 high regulatory T cells, thus resulting in a “suppression of the suppressors” [21]. A recent study in MS patients undergoing treatment with IFN-β showed that, in comparison to cells from healthy subjects, CD4+CD25 high T cells from untreated MS patients had increased mRNA for D1-like DR D5 and for TH. During treatment with IFN-β, both DR D5 and TH mRNA decreased down to values lower than those of cells from healthy controls. Most interestingly, in co-culture experiments, dopamine reduced the suppressive activity of CD4+CD25 high T cells from healthy subjects and completely abolished the suppressive activity of cells from untreated MS patients. On the contrary, in cells from MS patients treated with IFN-β for 12 months dopamine had no more effects on their suppressive ability [86].

    Such results likely indicate that in MS patients CD4+CD25 high T cells have increased ability to produce dopamine (as suggested by increased TH mRNA), in turn suppressing their regulatory function (as indicated by both increased D1-like DR D5 mRNA expression and ability of dopamine to completely abolish ex vivo their regulatory function). Increased (endogenous) dopamine-operated inhibition of CD4+CD25 high T cells likely contributes to the functional impairment of these cells in MS, resulting in enhanced disease activity [87,88], as also possibly suggested by the observation that TH mRNA levels after 12 months of IFN-β were significantly higher (on average 55%) in CD4+CD25 high T cells from patients experiencing clinical relapses in comparison to those without relapses during the study [86].

    From a general point of view, it seems that IFN-β induces a D1-like shift in immune cells of MS patients thus preparing these cells to respond to the potential antinflammatory action of endogenous dopamine and/or of dopaminergic agents (preferably selective for D1-like DRs). At the same time, IFN-β also reduces the D1-like DR-dependent (auto)-inhibitory loop on CD4+CD25 high T regulatory cells, thus reducing the possibility that dopaminergic stimulation may result in the functional inhibition of these cells, an effect likely detrimental in MS. Overall, evidence points to the evaluation of dopaminergic agonists as add-ons to IFN-β in the treatment of MS, as a strategy to increase its therapeutic efficacy. A detailed discussion of adrenergic and dopaminergic modulation of immunity in MS has been recently published [14].

    Endogenous production of catecholamines has been shown to occur also in inflamed synovial tissues of patients with rheumatoid arthritis (RA), where sympathetic innervation is reduced and local noradrenaline production is maintained by TH+ cells, mainly synovial macrophages (Miller et al., 2002). Noradrenaline levels correlate with the degree of inflammation and with spontaneous IL-8 secretion, while density of TH+ cells correlates positively with spontaneous secretion of IL-6, IL-8, and MMP-3 [89]. Further evidence for a critical role of local production of catecholamines by TH+ cells in RA synovium has been recently provided by showing that increased catecholamine release induced after blockade of VMAT2 with reserpine results in strong reduction of TNF (occurring through cAMP increase but possibly without involvement of classical β-ARs) and amelioration of inflammation in an animal model of RA [90]. Adrenergic mechanisms in the modulation of immune cells and their involvement in RA as well as in several other human diseases, including cancer, have been recently revised [14].

    6. Concluding remarks

    The ability of immune cells to synthesize and utilize dopamine, noradrenaline and adrenaline is now well established and significantly extends the well-known role of catecholamines in the neuroimmune cross-talk. Most importantly, intrinsic dopaminergic and adrenergic pathways in immune cells potentially represent an unprecedented opportunity for the specific and selective modulation of the immune response: indeed, the great number of potential pharmacological targets (including receptors, synthesizing and metabolising enzymes, transporters, etc.) is even exceeded by the availability of directly and indirectly acting pharmacological agents, already in clinical use for several non-immune indications and with a usually favourable therapeutic index (Tables 1-4). Dopaminergic and adrenergic agents actually represent an extremely attractive source of potentially novel immunomodulating agents with significant therapeutic potential.

    Nonetheless, several key points still need to be clarified, before it will be possible to exploit at best the immunomodulatory activity of endogenous (and exogenous) dopaminergic and adrenergic agents [13,14] (see also Figure 2).

    Figure 2. The cellular network sustained by endogenous catecholamines in human lymphocytes. Speculative scheme depicting the possible actions of endogenous catecholamines produced and released by human lymphocytes in the development of the immune response. CD4+CD25 high T regulatory cells (Treg) constitutively express TH, the key enzyme in the synthesis of catecholamines, DRs, α- and β-ARs, and contain high amounts of dopamine (DA), noradrenaline (NE), and adrenaline (E) stored in reserpine-sensitive compartments. Upon release, endogenous catecholamines (likely dopamine) subserve an autocrine/paracrine modulatory loop involving the activation of D1-like DR (D1/D5), leading to impaired suppressive activity of Treg towards mitogen-induced CD4+ T effector cells (Teff) proliferation. In the absence of stimulation, effector T lymphocytes express DRs, α- and β-ARs and contain trace amounts of DA, NE, and E. Upon stimulation, intracellular catecholamines increase by several ten-folds, and expression and function of both DRs and ARs undergo significant changes. Under these conditions, endogenous catecholamines may either directly affect cell survival and apoptosis from within the cell (lightnings), or they can be released (red arrows) to act upon lymphocytes themselves and/or upon neighbouring cells. Question marks (?) highlight various issues which await clarification. For the sake of clarity and simplicity, the picture does not include the potential role of catecholamines which are normally present into the extracellular space or which can be released from sympathoadrenergic terminals innervating lymphoid organs and tissues, or even which lymphocytes can encounter when they enter the brain in physiological (or pathological) situations. This figure was originally published in Blood. Cosentino M. et al. Human CD4+CD25+ regulatory T cells selectively express tyrosine hydroxylase and contain endogenous catecholamines subserving an autocrine/paracrine inhibitory functional loop. Blood. 2007 109:632-642. ©The American Society of Hematology.

    Outstanding questions include:

    – Catecholamine synthesis likely does not occur in all immune cells: which are the specific subsets and the functional conditions which trigger catecholamine production?

    – Does catecholamine production always include dopamine, noradrenaline and adrenaline or do “dopaminergic” and “adrenergic” immune cells actually exist?

    – Which are the catecholamine storage compartments and how is the releasing mechanism(s) regulated in the various immune cell populations? Which are the immune mediators stored and released together with catecholamines? Which are the physiological stimuli triggering catecholamine release from immune cells?

    – Adrenergic and dopaminergic receptors exist in multiple subtypes: are there cell subset-specific patterns of receptor expression? Which cell functions are controlled by each receptor in the various populations of immune cells?

    In addition, when dealing with the role of catecholaminergic pathways in immune cells in disease conditions, it is necessary to consider that:

    – receptor dysregulation occurring in disease states is not only specific for the receptor type but also for the cell subset(s)

    – receptors may be acted upon not only by exogenous but also by endogenous catecholamines directly produced by immune cells (Figure 5)

    – dynamic changes occur to receptor expression and responsiveness (and to endogenous catecholamine production) during treatment with immunomodulatory drugs (e.g., the case of IFN-β in MS).

    Last but not least, the pharmacological selectivity of adrenergic and dopaminergic agents must be carefully considered when choosing experimental drugs as well as when interpreting the resulting evidence: indeed, only well-constructed concentration-response curves and analysis of potency ratios of series of agonists and antagonists can provide consistent evidence for the involvement of specific receptor pathways.

    The origin itself of neuroimmunology can be traced back to the identification of sympathoadrenergic pathways as the main channel of communication between the nervous system and the immune system [91]. Interestingly, nowadays the discovery of endogenous catecholamines in immune cells requires an in-depth revision of the role of such mediators in the neuroimmune network, which will also likely provide novel and original approaches for targeted immunomodulation.

    Nonstandard Abbreviations:

    AR, adrenoceptor CNS, central nervous system COMT, catechol-O-methyl transferase DAO, d-amino acid oxidase DAT, dopamine transporter DBH, dopamine β-hydroxylase DR, dopaminergic receptor IFN, interferon IL, interleukin l-DOPA, l-3,4-dihydroxyphenylalanine LC, locus coeruleus LPS, lipopolysaccharide MAO, monoamine oxidase MS, multiple sclerosis NET, norepinephrine transporter NK, natural killer PBMC, peripheral blood mononuclear cell PHA, phytohemagglutinin PNMT, phenylethanolamine N-methyltransferase RA, rheumatoid arthritis TGF, transforming growth factor Th, T helper TH, tyrosine hydroxylase TNF, tumor necrosis factor VMAT, vesicular monoamine transporter.

    Cite this article as:
    Cosentino M, Kustrimovic N & F Marino, Endogenous catecholamines in immune cells: Discovery, functions and clinical potential as therapeutic targets (October 5, 2013). In BrainImmune: Trends in Neuroendocrine Immunology. Retrieved April 27, 2017, from http://brainimmune…link to the article’s URL here.

    Marco Cosentino, Natasa Kustrimovic, Franca Marino – Center for Research in Medical Pharmacology, University of Insubria, Varese, Italy
    Corresponding Author: Marco Cosentino, MD, PhD, E-mail: [email protected]

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    Contents

    Norepinephrine is a catecholamine and a phenethylamine. [4] Its structure differs from that of epinephrine only in that epinephrine has a methyl group attached to its nitrogen, whereas the methyl group is replaced by a hydrogen atom in norepinephrine. [4] The prefix nor- is derived as an abbreviation of the word "normal", used to indicate a demethylated compound. [5]

    Biosynthesis Edit

    Norepinephrine is synthesized from the amino acid tyrosine by a series of enzymatic steps in the adrenal medulla and postganglionic neurons of the sympathetic nervous system. While the conversion of tyrosine to dopamine occurs predominantly in the cytoplasm, the conversion of dopamine to norepinephrine by dopamine β-monooxygenase occurs predominantly inside neurotransmitter vesicles. [9] The metabolic pathway is:

    Phenylalanine → Tyrosine → L-DOPA → Dopamine → Norepinephrine [9]

    Thus the direct precursor of norepinephrine is dopamine, which is synthesized indirectly from the essential amino acid phenylalanine or the non-essential amino acid tyrosine. [9] These amino acids are found in nearly every protein and, as such, are provided by ingestion of protein-containing food, with tyrosine being the most common.

    Phenylalanine is converted into tyrosine by the enzyme phenylalanine hydroxylase, with molecular oxygen (O2) and tetrahydrobiopterin as cofactors. Tyrosine is converted into L-DOPA by the enzyme tyrosine hydroxylase, with tetrahydrobiopterin, O2, and probably ferrous iron (Fe 2+ ) as cofactors. [9] Conversion of tyrosine to L-DOPA is inhibited by Metyrosine, a tyrosine analog. L-DOPA is converted into dopamine by the enzyme aromatic L-amino acid decarboxylase (also known as DOPA decarboxylase), with pyridoxal phosphate as a cofactor. [9] Dopamine is then converted into norepinephrine by the enzyme dopamine β-monooxygenase (formerly known as dopamine β-hydroxylase), with O2 and ascorbic acid as cofactors. [9]

    Degradation Edit

    In mammals, norepinephrine is rapidly degraded to various metabolites. The initial step in the breakdown can be catalyzed by either of the enzymes monoamine oxidase (mainly monoamine oxidase A) or COMT. [10] From there the breakdown can proceed by a variety of pathways. The principal end products are either Vanillylmandelic acid or a conjugated form of MHPG, both of which are thought to be biologically inactive and are excreted in the urine. [11]

    Cellular effects Edit

    Adrenergic receptors in the mammal brain and body [11]
    Family Receptor Type Mechanism
    Alpha α1 Gq-coupled. Increase IP3 and calcium by
    activating phospholipase C.
    α2 Gi/Go-coupled. Decrease cAMP by
    inhibiting adenylate cyclase.
    Beta β1 Gs-coupled. Increase cAMP by
    activating adenylate cyclase.
    β2
    β3

    Like many other biologically active substances, norepinephrine exerts its effects by binding to and activating receptors located on the surface of cells. Two broad families of norepinephrine receptors have been identified, known as alpha and beta adrenergic receptors. [11] Alpha receptors are divided into subtypes α1 and α2 beta receptors into subtypes β1, β2, and β3. [11] All of these function as G protein-coupled receptors, meaning that they exert their effects via a complex second messenger system. [11] Alpha-2 receptors usually have inhibitory effects, but many are located pre-synaptically (i.e., on the surface of the cells that release norepinephrine), so the net effect of alpha-2 activation is often a decrease in the amount of norepinephrine released. [11] Alpha-1 receptors and all three types of beta receptors usually have excitatory effects. [11]

    Storage, release, and reuptake Edit

    Inside the brain norepinephrine functions as a neurotransmitter, and is controlled by a set of mechanisms common to all monoamine neurotransmitters. After synthesis, norepinephrine is transported from the cytosol into synaptic vesicles by the vesicular monoamine transporter (VMAT). [12] VMAT can be inhibited by Reserpine causing a decrease in neurotransmitter stores. Norepinephrine is stored in these vesicles until it is ejected into the synaptic cleft, typically after an action potential causes the vesicles to release their contents directly into the synaptic cleft through a process called exocytosis. [11]

    Once in the synapse, norepinephrine binds to and activates receptors. After an action potential, the norepinephrine molecules quickly become unbound from their receptors. They are then absorbed back into the presynaptic cell, via reuptake mediated primarily by the norepinephrine transporter (NET). [13] Once back in the cytosol, norepinephrine can either be broken down by monoamine oxidase or repackaged into vesicles by VMAT, making it available for future release. [12]

    Sympathetic nervous system Edit

    Norepinephrine is the main neurotransmitter used by the sympathetic nervous system, which consists of about two dozen sympathetic chain ganglia located next to the spinal cord, plus a set of prevertebral ganglia located in the chest and abdomen. [14] These sympathetic ganglia are connected to numerous organs, including the eyes, salivary glands, heart, lungs, liver, gallbladder, stomach, intestines, kidneys, urinary bladder, reproductive organs, muscles, skin, and adrenal glands. [14] Sympathetic activation of the adrenal glands causes the part called the adrenal medulla to release norepinephrine (as well as epinephrine) into the bloodstream, from which, functioning as a hormone, it gains further access to a wide variety of tissues. [14]

    Broadly speaking, the effect of norepinephrine on each target organ is to modify its state in a way that makes it more conducive to active body movement, often at a cost of increased energy use and increased wear and tear. [15] This can be contrasted with the acetylcholine-mediated effects of the parasympathetic nervous system, which modifies most of the same organs into a state more conducive to rest, recovery, and digestion of food, and usually less costly in terms of energy expenditure. [15]

    The sympathetic effects of norepinephrine include:

    • In the eyes, an increase in production of tears, making the eyes more moist, [16] and pupil dilation through contraction of the iris dilator.
    • In the heart, an increase in the amount of blood pumped. [17]
    • In brown adipose tissue, an increase in calories burned to generate body heat (thermogenesis). [18]
    • Multiple effects on the immune system. The sympathetic nervous system is the primary path of interaction between the immune system and the brain, and several components receive sympathetic inputs, including the thymus, spleen, and lymph nodes. However the effects are complex, with some immune processes activated while others are inhibited. [19]
    • In the arteries, constriction of blood vessels, causing an increase in blood pressure. [20]
    • In the kidneys, release of renin and retention of sodium in the bloodstream. [21]
    • In the liver, an increase in production of glucose, either by glycogenolysis after a meal or by gluconeogenesis when food has not recently been consumed. [21] Glucose is the body's main energy source in most conditions.
    • In the pancreas, increased release of glucagon, a hormone whose main effect is to increase the production of glucose by the liver. [21]
    • In skeletal muscles, an increase in glucose uptake. [21]
    • In adipose tissue (i.e., fat cells), an increase in lipolysis, that is, conversion of fat to substances that can be used directly as energy sources by muscles and other tissues. [21]
    • In the stomach and intestines, a reduction in digestive activity. This results from a generally inhibitory effect of norepinephrine on the enteric nervous system, causing decreases in gastrointestinal mobility, blood flow, and secretion of digestive substances. [22]

    Noradrenaline and ATP are sympathetic co-transmitters. It is found that the endocannabinoid anandamide and the cannabinoid WIN 55,212-2 can modify the overall response to sympathetic nerve stimulation, which indicates that prejunctional CB1 receptors mediate the sympatho-inhibitory action. Thus cannabinoids can inhibit both the noradrenergic and purinergic components of sympathetic neurotransmission. [23]

    Central nervous system Edit

    The noradrenergic neurons in the brain form a neurotransmitter system, that, when activated, exerts effects on large areas of the brain. The effects are manifested in alertness, arousal, and readiness for action.

    Noradrenergic neurons (i.e., neurons whose primary neurotransmitter is norepinephrine) are comparatively few in number, and their cell bodies are confined to a few relatively small brain areas, but they send projections to many other brain areas and exert powerful effects on their targets. These noradrenergic cell groups were first mapped in 1964 by Annica Dahlström and Kjell Fuxe, who assigned them labels starting with the letter "A" (for "aminergic"). [24] In their scheme, areas A1 through A7 contain the neurotransmitter norepinephrine (A8 through A14 contain dopamine). Noradrenergic cell group A1 is located in the caudal ventrolateral part of the medulla, and plays a role in the control of body fluid metabolism. [25] Noradrenergic cell group A2 is located in a brainstem area called the solitary nucleus these cells have been implicated in a variety of responses, including control of food intake and responses to stress. [26] Cell groups A5 and A7 project mainly to the spinal cord. [27]

    The most important source of norepinephrine in the brain is the locus coeruleus, which contains noradrenergic cell group A6 and adjoins cell group A4. The locus coeruleus is quite small in absolute terms—in primates it is estimated to contain around 15,000 neurons, less than one-millionth of the neurons in the brain—but it sends projections to every major part of the brain and also to the spinal cord. [28]

    The level of activity in the locus coeruleus correlates broadly with vigilance and speed of reaction. LC activity is low during sleep and drops to virtually nothing during the REM (dreaming) state. [29] It runs at a baseline level during wakefulness, but increases temporarily when a person is presented with any sort of stimulus that draws attention. Unpleasant stimuli such as pain, difficulty breathing, bladder distension, heat or cold generate larger increases. Extremely unpleasant states such as intense fear or intense pain are associated with very high levels of LC activity. [28]

    Norepinephrine released by the locus coeruleus affects brain function in a number of ways. It enhances processing of sensory inputs, enhances attention, enhances formation and retrieval of both long term and working memory, and enhances the ability of the brain to respond to inputs by changing the activity pattern in the prefrontal cortex and other areas. [30] The control of arousal level is strong enough that drug-induced suppression of the LC has a powerful sedating effect. [29]

    There is great similarity between situations that activate the locus coeruleus in the brain and situations that activate the sympathetic nervous system in the periphery: the LC essentially mobilizes the brain for action while the sympathetic system mobilizes the body. It has been argued that this similarity arises because both are to a large degree controlled by the same brain structures, particularly a part of the brainstem called the nucleus gigantocellularis. [28]

    Skin Edit

    Norepinephrine is also produced by Merkel cells which are part of the somatosensory system. It activates the afferent sensory neuron. [31]

    A large number of important drugs exert their effects by interacting with norepinephrine systems in the brain or body. Their uses include treatment of cardiovascular problems, shock, and a variety of psychiatric conditions. These drugs are divided into: sympathomimetic drugs which mimic or enhance at least some of the effects of norepinephrine released by the sympathetic nervous system sympatholytic drugs, in contrast, block at least some of the effects. [32] Both of these are large groups with diverse uses, depending on exactly which effects are enhanced or blocked. [32]

    Norepinephrine itself is classified as a sympathomimetic drug: its effects when given by intravenous injection of increasing heart rate and force and constricting blood vessels make it very useful for treating medical emergencies that involve critically low blood pressure. [32] Surviving Sepsis Campaign recommended norepinephrine as first line agent in treating septic shock which is unresponsive to fluid resuscitation, supplemented by vasopressin and epinephrine. Dopamine usage is restricted only to highly selected patients. [33]

    Beta blockers Edit

    These are sympatholytic drugs that block the effects of beta adrenergic receptors while having little or no effect on alpha receptors. They are sometimes used to treat high blood pressure, atrial fibrillation and congestive heart failure, but recent reviews have concluded that other types of drugs are usually superior for those purposes. [34] [35] Beta blockers may be a viable choice for other cardiovascular conditions, though, including angina and Marfan syndrome. [36] They are also widely used to treat glaucoma, most commonly in the form of eyedrops. [37] Because of their effects in reducing anxiety symptoms and tremor, they have sometimes been used by entertainers, public speakers and athletes to reduce performance anxiety, although they are not medically approved for that purpose and are banned by the International Olympic Committee. [38] [39]

    However, the usefulness of beta blockers is limited by a range of serious side effects, including slowing of heart rate, a drop in blood pressure, asthma, and reactive hypoglycemia. [37] The negative effects can be particularly severe in people who suffer from diabetes. [34]

    Alpha blockers Edit

    These are sympatholytic drugs that block the effects of adrenergic alpha receptors while having little or no effect on beta receptors. [40] Drugs belonging to this group can have very different effects, however, depending on whether they primarily block alpha-1 receptors, alpha-2 receptors, or both. Alpha-2 receptors, as described elsewhere in this article, are frequently located on norepinephrine-releasing neurons themselves and have inhibitory effects on them consequently, blockage of alpha-2 receptors usually results in an increase in norepinephrine release. [40] Alpha-1 receptors are usually located on target cells and have excitatory effects on them consequently, blockage of alpha-1 receptors usually results in blocking some of the effects of norepinephrine. [40] Drugs such as phentolamine that act on both types of receptors can produce a complex combination of both effects. In most cases when the term "alpha blocker" is used without qualification, it refers to a selective alpha-1 antagonist.

    Selective alpha-1 blockers have a variety of uses. Since one of their effects is to inhibit the contraction of the smooth muscle in the prostate, they are often used to treat symptoms of benign prostatic hyperplasia. [41] Alpha-blockers also likely help people pass their kidney stones. [42] Their effects on the central nervous system make them useful for treating generalized anxiety disorder, panic disorder, and posttraumatic stress disorder. [43] They may, however, have significant side-effects, including a drop in blood pressure. [40]

    Some antidepressants function partly as selective alpha-2 blockers, but the best-known drug in that class is yohimbine, which is extracted from the bark of the African yohimbe tree. [44] Yohimbine acts as a male potency enhancer, but its usefulness for that purpose is limited by serious side-effects including anxiety and insomnia. [44] Overdoses can cause a dangerous increase in blood pressure. [44] Yohimbine is banned in many countries, but in the United States, because it is extracted from a plant rather than chemically synthesized, it is sold over the counter as a nutritional supplement. [45]

    Alpha-2 agonists Edit

    These are sympathomimetic drugs that activate alpha-2 receptors or enhance their effects. [46] Because alpha-2 receptors are inhibitory and many are located presynaptically on norepinephrine-releasing cells, the net effect of these drugs is usually to reduce the amount of norepinephrine released. [46] Drugs in this group that are capable of entering the brain often have strong sedating effects, due to their inhibitory effects on the locus coeruleus. [46] Clonidine, for example, is used for the treatment of anxiety disorders and insomnia, and also as a sedative premedication for patients about to undergo surgery. [47] Xylazine, another drug in this group, is also a powerful sedative and is often used in combination with ketamine as a general anaesthetic for veterinary surgery—in the United States it has not been approved for use in humans. [48]

    Stimulants and antidepressants Edit

    These are drugs whose primary effects are thought to be mediated by different neurotransmitter systems (dopamine for stimulants, serotonin for antidepressants), but many also increase levels of norepinephrine in the brain. [49] Amphetamine, for example, is a stimulant that increases release of norepinephrine as well as dopamine. [50] Monoamine oxidase inhibitors are antidepressants that inhibit the metabolic degradation of norepinephrine as well as serotonin and dopamine. [51] In some cases it is difficult to distinguish the norepinephrine-mediated effects from the effects related to other neurotransmitters. [ citation needed ]

    A number of important medical problems involve dysfunction of the norepinephrine system in the brain or body.

    Sympathetic hyperactivation Edit

    Hyperactivation of the sympathetic nervous system is not a recognized condition in itself, but it is a component of a number of conditions, as well as a possible consequence of taking sympathomimetic drugs. It causes a distinctive set of symptoms including aches and pains, rapid heartbeat, elevated blood pressure, sweating, palpitations, anxiety, headache, paleness, and a drop in blood glucose. If sympathetic activity is elevated for an extended time, it can cause weight loss and other stress-related body changes.

    The list of conditions that can cause sympathetic hyperactivation includes severe brain injury, [52] spinal cord damage, [53] heart failure, [54] high blood pressure, [55] kidney disease, [56] and various types of stress.

    Pheochromocytoma Edit

    A pheochromocytoma is a rarely occurring tumor of the adrenal medulla, caused either by genetic factors or certain types of cancer. The consequence is a massive increase in the amount of norepinephrine and epinephrine released into the bloodstream. The most obvious symptoms are those of sympathetic hyperactivation, including particularly a rise in blood pressure that can reach fatal levels. The most effective treatment is surgical removal of the tumor.

    Stress Edit

    Stress, to a physiologist, means any situation that threatens the continued stability of the body and its functions. [57] Stress affects a wide variety of body systems: the two most consistently activated are the hypothalamic-pituitary-adrenal axis and the norepinephrine system, including both the sympathetic nervous system and the locus coeruleus-centered system in the brain. [57] Stressors of many types evoke increases in noradrenergic activity, which mobilizes the brain and body to meet the threat. [57] Chronic stress, if continued for a long time, can damage many parts of the body. A significant part of the damage is due to the effects of sustained norepinephrine release, because of norepinephrine's general function of directing resources away from maintenance, regeneration, and reproduction, and toward systems that are required for active movement. The consequences can include slowing of growth (in children), sleeplessness, loss of libido, gastrointestinal problems, impaired disease resistance, slower rates of injury healing, depression, and increased vulnerability to addiction. [57]

    ADHD Edit

    Attention deficit hyperactivity disorder is a psychiatric condition involving problems with attention, hyperactivity, and impulsiveness. [58] It is most commonly treated using stimulant drugs such as methylphenidate (Ritalin), whose primary effect is to increase dopamine levels in the brain, but drugs in this group also generally increase brain levels of norepinephrine, and it has been difficult to determine whether these actions are involved in their clinical value. There is also substantial evidence that many people with ADHD show biomarkers involving altered norepinephrine processing. [59] Several drugs whose primary effects are on norepinephrine, including guanfacine, clonidine, and atomoxetine, have been tried as treatments for ADHD, and found to have effects comparable to those of stimulants. [60] [61]

    Autonomic failure Edit

    Several conditions, including Parkinson's disease, diabetes and so-called pure autonomic failure, can cause a loss of norepinephrine-secreting neurons in the sympathetic nervous system. The symptoms are widespread, the most serious being a reduction in heart rate and an extreme drop in resting blood pressure, making it impossible for severely affected people to stand for more than a few seconds without fainting. Treatment can involve dietary changes or drugs. [62]

    Norepinephrine has been reported to exist in a wide variety of animal species, including protozoa, [63] placozoa and cnidaria (jellyfish and related species), [64] but not in ctenophores (comb jellies), whose nervous systems differ greatly from those of other animals. [65] It is generally present in deuterostomes (vertebrates, etc.), but in protostomes (arthropods, molluscs, flatworms, nematodes, annelids, etc.) it is replaced by octopamine, a closely related chemical with a closely related synthesis pathway. [63] In insects, octopamine has alerting and activating functions that correspond (at least roughly) with the functions of norepinephrine in vertebrates. [66] It has been argued that octopamine evolved to replace norepinephrine rather than vice versa however, the nervous system of amphioxus (a primitive chordate) has been reported to contain octopamine but not norepinephrine, which presents difficulties for that hypothesis. [63]

    Early in the twentieth century Walter Cannon, who had popularized the idea of a sympathoadrenal system preparing the body for fight and flight, and his colleague Arturo Rosenblueth developed a theory of two sympathins, sympathin E (excitatory) and sympathin I (inhibitory), responsible for these actions. [67] The Belgian pharmacologist Zénon Bacq as well as Canadian and US-American pharmacologists between 1934 and 1938 suggested that noradrenaline might be a sympathetic transmitter. [67] In 1939, Hermann Blaschko and Peter Holtz independently identified the biosynthetic mechanism for norepinephrine in the vertebrate body. [68] [69] In 1945 Ulf von Euler published the first of a series of papers that established the role of norepinephrine as a neurotransmitter. [70] He demonstrated the presence of norepinephrine in sympathetically innervated tissues and brain, and adduced evidence that it is the sympathin of Cannon and Rosenblueth. Stanley Peart was the first to demonstrate the release of noradrenaline after the stimulation of sympathetic nerves.


    Can catecholamines degrade back into tyrosine, or, is synthesis irreversible? (in human body) - Biology

    Nature uses a diverse spectrum of molecules as hormones, and knowing the basic structure of a hormone imparts considerable knowledge about its receptor and mechanism of action. Additionally, the simpler structures can often be exploited to generate similar molecules - agonists and antagonists - that are therapeutically valuable.

    Like all molecules, hormones are synthesized, exist in a biologically active state for a time, and then degrade or are destroyed. Again, having an appreciation for the "halflife" and mode of elimination of a hormone aids in understanding its role in physiology and is critical when using hormones as drugs.

    Most commonly, hormones are categorized into four structural groups, with members of each group having many properties in common:

    • Peptides and proteins
    • Steroids
    • Amino acid derivatives
    • Fatty acid derivatives - Eicosanoids

    Peptides and Proteins

    Peptide and protein hormones are, of course, products of translation. They vary considerably in size and post-translational modifications, ranging from peptides as short as three amino acids to large, multisubunit glycoproteins.

    Many protein hormones are synthesized as prohormones, then proteolytically clipped to generate their mature form. In other cases, the hormone is originally embedded within the sequence of a larger precursor, then released by multiple proteolytic cleavages.

    Peptide hormones are synthesized in endoplasmic reticulum, transferred to the Golgi and packaged into secretory vesicles for export. They can be secreted by one of two pathways:

    • Regulated secretion: The cell stores hormone in secretory granules and releases them in "bursts" when stimulated. This is the most commonly used pathway and allows cells to secrete a large amount of hormone over a short period of time.
    • Constitutive secretion: The cell does not store hormone, but secretes it from secretory vesicles as it is synthesized.

    Most peptide hormones circulate unbound to other proteins, but exceptions exist for example, insulin-like growth factor-1 binds to one of several binding proteins. In general, the halflife of circulating peptide hormones is only a few minutes.

    Steroids

    Steroids are lipids and, more specifically, derivatives of cholesterol. Examples include the sex steroids such as testosterone and adrenal steroids such as cortisol.

    The first and rate-limiting step in the synthesis of all steroid hormones is conversion of cholesterol to pregnenolone, which is illustrated here to demonstate the system of numbering rings and carbons for identification of different steroid hormones.

    Pregnenolone is formed on the inner membrane of mitochondria then shuttled back and forth between mitochondrion and the endoplasmic reticulum for further enzymatic transformations involved in synthesis of derivative steroid hormones.

    Newly synthesized steroid hormones are rapidly secreted from the cell, with little if any storage. Increases in secretion reflect accelerated rates of synthesis. Following secretion, all steroids bind to some extent to plasma proteins. This binding is often low affinity and non-specific (e.g. to albumin), but some steroids are transported by specific binding proteins, which clearly affects their halflife and rate of elimination.

    Steroid hormones are typically eliminated by inactivating metabolic transformations and excretion in urine or bile.

    Amino Acid Derivatives

    There are two groups of hormones derived from the amino acid tyrosine:

    • Thyroid hormones are basically a "double" tyrosine with the critical incorporation of 3 or 4 iodine atoms.
    • Catecholamines include epinephrine and norepinephrine, which are used as both hormones and neurotransmitters.

    The pathways to synthesis of these hormones is provided in the sections on the thyroid gland and the adrenal medulla.

    The circulating halflife of thyroid hormones is on the order of a few days. They are inactivated primarily by intracellular deiodinases. Catecholamines, on the other hand, are rapidly degraded, with circulating halflives of only a few minutes.

    Two other amino acids are used for synthesis of hormones:

    • Tryptophan is the precursor to serotonin and the pineal hormone melatonin
    • Histidine is converted to histamine

    Fatty Acid Derivatives - Eicosanoids

    Eicosanoids are a large group of molecules derived from polyunsaturated fatty acids. The principal groups of hormones of this class are prostaglandins, prostacyclins, leukotrienes and thromboxanes.

    Arachadonic acid is the most abundant precursor for these hormones. Stores of arachadonic acid are present in membrane lipids and released through the action of various lipases. The specific eicosanoids synthesized by a cell are dictated by the battery of processing enzymes expressed in that cell.

    These hormones are rapidly inactivated by being metabolized, and are typically active for only a few seconds.

    Hormones, Receptors and Target Cells

    Control of Endocrine Activity


    Human tyrosine hydroxylase in Parkinson’s disease and in related disorders

    Parkinson’s disease (PD) is an aging-related movement disorder mainly caused by a deficiency of neurotransmitter dopamine (DA) in the striatum of the brain and is considered to be due to progressive degeneration of nigro-striatal DA neurons. Most PD is sporadic without family history (sPD), and there are only a few percent of cases of young-onset familial PD (fPD, PARKs) with the chromosomal locations and the genes identified. Tyrosine hydroxylase (TH), tetrahydrobiopterin (BH4)-dependent and iron-containing monooxygenase, catalyzes the conversion of l -tyrosine to l -3,4-dihydroxyphenylalanine ( l -DOPA), which is the initial and rate-limiting step in the biosynthesis of catecholamines (DA, noradrenaline, and adrenaline). PD affects specifically TH-containing catecholamine neurons. The most marked neurodegeneration in patients with DA deficiency is observed in the nigro-striatal DA neurons, which contain abundant TH. Accordingly, TH has been speculated to play some important roles in the pathophysiology in PD. However, this decrease in TH is thought to be secondary due to neurodegeneration of DA neurons caused by some as yet unidentified genetic and environmental factors, and thus, TH deficiency may not play a direct role in PD. This manuscript provides an overview of the role of human TH in the pathophysiology of PD, covering the following aspects: (1) structures of the gene and protein of human TH in relation to PD (2) similarity and dissimilarity between the phenotypes of aging-related sPD and those of young-onset fPD or DOPA-responsive dystonia due to DA deficiency in the striatum with decreased TH activity caused by mutations in either the TH gene or GTP cyclohydrolase I (GCH1) gene and (3) genetic variants of the TH gene (polymorphisms, rare variants, and mutations) in PD, as discovered recently by advanced genome analysis.

    This is a preview of subscription content, access via your institution.


    Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9.figshare.c.3744362.

    Published by the Royal Society. All rights reserved.

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    JM and DW drafted and wrote the manuscript. KH contributed to the critical revision of the manuscript. DW prepared Figures ​ Figures1, 1 , ​ ,2 2 and ​ and5 5 JM and DW prepared Figures ​ Figures3 3 and ​ and4. 4 . All authors read and approved the final manuscript.

    The Authors want to thank the Metabolomics group of the LCSB for careful reading and feedback. Related research of the authors was supported by the Fonds National de la Recherche (FNR) Luxembourg (ATTRACT A10/03 and AFR-Postdoc-3973022).


    Watch the video: Metabolism of Catecholamines Dopamine, Noradrenaline and Adrenaline (July 2022).


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