Catecholamine biosynthesis and physiological regulation

in neuroendocrine cells

T . F L A T M A R K

Department of Biochemistry and Molecular Biology, University of Bergen, Bergen, Norway

ABSTRACT

The catecholamines are widely distributed in mammals and their levels and physiological functions

are regulated at many sites. These include their release from neuroendocrine cells, the type and

sensitivity of the multiple receptors in target cells, the efficacy of the reuptake system in the

secretory cells, and the rates of catecholamine biosynthesis and degradation. In the present review

the main focus will be on the more recent studies on the biosynthesis in neuroendocrine cells which

involves a specific set of enzymes, with special reference to physiologically important regulatory

mechanisms. Eight enzymes of the biosynthetic pathway have now been identified, cloned,

expressed as recombinant proteins, characterized with respect to catalytic and regulatory properties,

and some of them also crystallized. The identification of the tyrosine hydroxylase catalysed reaction

as the rate-limiting step in the normal catecholamine biosynthesis has attracted most attention, both

in terms of transcriptional and post-translational regulation. In certain human genetic disorders of

catecholamine biosynthesis other enzymes in the pathway may become rate-limiting, notably those

involved in the biosynthesis/regeneration of the natural co-factor tetrahydrobiopterin in the tyrosine

hydroxylase reaction. The enzymes involved seem to be regulated by a variety of physiological

factors, both on a long-term scale and a short-term basis, and include the relative rates of synthesis,

degradation and state of activation of the biosynthetic enzymes, notably of tyrosine hydroxylase.

Multiple surface receptors and signalling pathways are activated in response to extracellular stimuli

and play an essential role in the regulation of catecholamine biosynthesis.

Keywords adrenaline, biosynthesis, catecholamine, dopamine, human genetics, L-DOPA,

noradrenaline, post-translational regulation, tetrahydrobiopterin, transcriptional regula-

tion, transgenic animals.

Received 29 March 1999, accepted 28 June 1999

Chemical neurotransmission as a concept is generally

attributed to Elliott (1904, 1905), who emphasized the

similarity between the action of adrenaline and

sympathetic nerve stimulation and thereby transformed

an intuitive feeling about neurotransmission into a

scienti®c working hypothesis. Loewi (1921) provided

the experimental proof for the chemical nature of

neurotransmission in the frog, identifying the active

principle correctly as adrenaline (A). That noradrenaline

is the sympathetic neurotransmitter in mammals and

most other animals was established by von Euler

(1946), who identi®ed NA in the splenic nerves, and

organs supplied by these nerves, by chemical isolation.

Shortly after that, NA was found to be a normal

constituent of the adrenal gland and mammalian brain

(Holtz 1950) which laid the foundation for a new area

of research in the ®eld of catecholamines (CAs) and

chemical neurotransmission in general. In addition to

these fundamental discoveries, von Euler also correctly

predicted that NA was highly concentrated in the nerve

terminal region from which it was released to act as a

neurotransmitter. This prediction was conclusively

documented with the ®rst isolated fraction of NA-

storing vesicles from sympathetic nerves (von Euler &

Hillarp 1957) and further con®rmed with the devel-

opment of the ¯uorescence histochemical technique

(Falch et al. 1962). Sympathetic ganglia were also

found to contain NA in a concentration similar to that

found in the post-ganglionic ®bres. The correlation

between the NA content of a nerve or an organ and its

content of adrenergic ®bres is now so well established

that the occurrence of NA in a given organ or nerve

can, in general, be taken as evidence for the presence of

adrenergic ®bres provided the presence of chromaf®n

Correspondence: Prof. Torgeir Flatmark, Department of Biochemistry and Molecular Biology, University of Bergen, AÊ rstadveien 19, N-5009

Bergen, Norway.

Acta Physiol Scand 2000, 168, 1±17

Ó 2000 Scandinavian Physiological Society 1

tissue can be excluded (von Euler 1971). The ubiqui-

tous distribution of NA in tissues is consistent with the

presence of adrenergic vasomotor ®bres in almost all

peripheral tissues. The central nervous system (CNS) of

mammals contains all three CAs (DA, NA and A),

localized to speci®c areas of the brain. Detailed maps of

the pattern of distribution of all the CAs in the CNS

have been obtained for different animal species, as ®rst

demonstrated for NA/A (Vogt 1954). Systematic

studies on the regional and laminar distribution of

catecholaminergic ®bres in the human cerebral cortex

have demonstrated major evolutionary changes in the

organization of the cortical monoaminergic input as

compared with rodents (Gaspar et al. 1989). DA

represents about 50% of the total CA content in the

CNS of most mammals, and in addition to being a

biosynthetic precursor of NA and A it has an important

transmitter function (Carlsson 1959). The presence of

abundant DA, e.g. in the basal ganglia, has stimulated

intensive research on the functional aspects of this

compound, and it is now well established that this

monoamine has an important role in extrapyramidal

function. In addition to its function as the principal

neurotransmitter of the sympathetic nervous system

NA is a major CNS transmitter (Bloom & Hoffer

1973). In contrast, the concentration of A is relatively

low, 7±14% of the NA content (Vogt 1954). A is widely

distributed in the brains of various species throughout

phylogeny, but is generally localized to the hypothal-

amus and brainstem/medulla in all species studied

(Mefford 1988). Immuno-histochemical evidence has

been presented for the existence of A neurones in the

rat brain (HoÈkfelt et al. 1974). The present model

suggests that A formed in these neurones is primarily a

co-transmitter with NA formed in the same terminals,

notably in the brainstem/medulla (Mefford 1987)

whereas in the hypothalamus A appears to be primarily

a hormone/paracrine regulator (Mefford 1988).

In contrast to the effects of acetylcholine (ACh) the

actions of CAs are not limited to the site of release but

are more diffuse, typically extending beyond an indi-

vidual target. Some NA released from nerve terminals

escapes reuptake and enters the circulation (Peart 1949,

Brown & Gillespie 1957). Thus, sympathetic ganglia are

the major contributors to the stress-elicited rise in

circulating NA (Nankova et al. 1996). In addition, the

adrenal medulla releases A and NA into the circulation.

This diffuse release of CAs, along with the divergent

anatomical organization of sympathetic pre-ganglionic

axons (Loewy & Spyer 1990), underlies the ability of

the sympathetic nervous system to achieve a concerted

hormone-like activation of sympathetic targets in situ-

ations of stress.

The concept of a third, peripheral catecholamin-

ergic system has been proposed more recently

(Goldstein et al. 1995), where DA derived from

plasma 3,4-dihydroxy-L-phenylalanine (L-DOPA) acts

as an autocrine/paracrine regulator of local organ

function (e.g. in kidneys, gastric mucosa, lungs and

mesenteric organs). Generation of DA in non-

adrenergic cells may explain why human urine

contains higher concentrations of DA and its

metabolites (DA is inactivated by conjugation to

sulphate) than of NA and its metabolites.

During the past decades great advances have been

made in the understanding of the biochemistry, cell

biology, molecular biology, genetics, physiology, clinical

importance and pharmacology of catecholaminergic

systems. This is not unexpected considering the fact

that CAs in¯uence the functions of such a wide variety

of tissues. The progress is based on the development of

new in vitro and in vivo experimental approaches and

technologies. Examples are radioimmunoassays, ¯uo-

rescence histochemistry, electron microscopy, cell and

subcellular fractionation, ultrasensitive chromato-

graphic methods (HPLC and GC-MS), capillary elec-

trophoresis, i.e. methods for detection/visualizing/

quantitating CAs and their metabolic enzymes, trans-

porters and receptors at the cellular and subcellular

level, both in vitro and in vivo. At this point the molecular

biology of the catecholaminergic neuroendocrine cells

is also well understood and key enzymes, transporters

and receptors related to CA biosynthesis, storage/

release and function have been cloned, mutated,

expressed as recombinant proteins and characterized in

terms of structure and function, and some also crys-

tallized. All these developments represent the basis for

the current state of knowledge on the physiological

regulation of CA functions. Most of this review will

refer to the progress made during the last decade in our

understanding of the pathways for the biosynthesis of

CAs and their physiological regulation in neuroendo-

crine cells. For a recent historic review on catechol-

aminergic neurotransmission, see StjaÈrne (1999).

FUNCTIONAL ASSESSMENT OF THE

CATECHOLAMINERGIC SYSTEMS

Sympathoadrenal activity can be assessed by measuring

CAs in plasma or by recording impulses in sympathetic

nerves to skin and muscles by microneurography

(Christensen 1991, Hjemdahl 1993). Numerous meth-

odologies have been published for the assay of CAs in

plasma, illustrating the fact that there are many ana-

lytical problems (Rosano et al. 1991, Hjemdahl 1993).

However, when correctly sampled, measured and

interpreted, plasma CAs can yield very valuable infor-

mation on sympathoadrenal activity.

Information about the activity of the central cate-

cholaminergic systems can be obtained by assay of CAs

Regulation of catecholamine biosynthesis � T Flatmark Acta Physiol Scand 2000, 168, 1±17

2 Ó 2000 Scandinavian Physiological Society

and their metabolites as well as the co-factor tetrahy-

drobiopterin (BH4) and related metabolites in the

cerebrospinal ¯uid (CSF). Such analyses have been

particularly informative in studies on transgenic models

and human genetic disorders of CA metabolism (see

below). Thus, impaired hydroxylation of L-tyrosine

(L-Tyr) owing to de®ciency of functional tyrosine

hydroxylase (TH) or to a de®ciency in the supply of its

co-factor BH4 reduces the formation of CAs.

Furthermore, a de®ciency of functional dopamine

b-hydroxylase (DBH) results in a more speci®c accu-

mulation of L-DOPA and DA and decreased synthesis

of NA/A (see below). The measurement of respective

metabolites in the CSF is of capital importance for the

clinical diagnosis and treatment follow-up of the human

genetic disorders of CA metabolism.

Rapid progress in effective methods to image brain

functions has revolutionized neuroscience. It is now

possible to study non-invasively in humans neural

processes that were previously only accessible in

experimental animals and in brain-injured patients. In

order to assess the function of e.g. the nigrostriatal

dopaminergic system in vivo quantitative dynamic posi-

tron emission tomography (PET) with the L-DOPA

analogue 3,4-dihydroxy-6-[18F]¯uoro-L-phenylalanine

([18F]FDOPA) as tracer is now a commonly used

experimental and clinical approach, e.g. in the clinical

assessment of patients with movement disorders

including idiopathic Parkinsonism (Parkinson's disease)

(Eidelberg 1992, Cumming & Gjedde 1998). This

technique of functional neuroimaging has also provided

important in vivo insights into the nigrostriatal dopa-

minergic system (Eidelberg 1992) and has been shown

to be a valuable method to estimate, e.g. the 3,4-dihy-

droxy-L-phenylalanine (L-DOPA) decarboxylase (DDC)

activity and to study potential regulatory properties of

the enzyme in vivo. As will be discussed below, recent

advances in genetic manipulation of the mouse have

also been very informative in the functional assessment

of the catecholaminergic systems.

CATECHOLAMINE BIOSYNTHETIC

PATHWAY

The term catecholamine (CA) refers, generally, to all

organic compounds that contain a catechol nucleus

(a benzene ring with two adjacent hydroxyl substitu-

ents) and an amine group. In a physiological context

CA implies 3,4-dihydrophenylethylamine (dopamine,

DA) and its metabolic products, noradrenaline (NA)

and adrenaline (A). These CAs are synthesized from the

amino acid L-tyrosine (L-Tyr) in a common biosynthetic

pathway that uses six enzymes: tyrosine hydroxylase

(TH), pterin-4a-carbinolamine dehydratase (PCD),

dihyropteridine reductase (DHPR), 3,4-dihydroxy-

L-phenylalanine (L-DOPA) decarboxylase (DDC),

dopamine b-hydroxylase (DBH), and phenylethylamine

N-methyl transferase (PNMT) (Figs 1 and 2). In addi-

tion, as seen from Fig. 2 this biosynthetic pathway is

dependent on an adequate supply of the speci®c co-

factor tetrahydrobiopterin [(6R)-L-erythro-5,6,7,8-tetra-

hydrobiopterin, BH4) which is synthesized from GTP

by three consecutive enzyme steps as well as ascorbate

which functions as an electron donor in the hydroxyl-

ation of DA to NA (Fig. 3). During the last few years

considerable progress has been made in the elucidation

of these metabolic pathways of CA biosynthesis,

including catalytic and regulatory properties as well as

crystal structure analysis of most of the enzymes

involved.

Tyrosine hydroxylase

CAs are synthesized from their amino acid precursor

L-Tyr by a sequence of enzymatic steps (Figs 1 and 2)

®rst postulated by Blaschko (1939) and ®nally

con®rmed by Nagatsu et al. (1964) when they demon-

strated that the enzyme tyrosine hydroxylase (TH,

tyrosine 3-monoxygenase, EC 1.14.16.2) is responsible

for the conversion of L-Tyr to L-DOPA. The conver-

sion of L-Tyr to NA and A was ®rst shown in the

adrenal medulla (Blaschko 1939), and later con®rmed in

brain, sympathetic ganglia, sympathetic nerves and in all

sympathetically innervated tissues studied to date. The

enzyme is a speci®c phenotypic marker of CA produ-

cing cells in the CNS and PNS.

TH requires Fe(II) at the active site, tetrahydro-

biopterin co-factor and dioxygen (Fig. 2), and shows a

fairly high degree of substrate speci®city; it hydroxylates

L-Tyr (stereospeci®c) and to a smaller extent

L-phenylalanine (Fukami et al. 1990). The enzyme is

present in the neuroendocrine cells both in a soluble

and a membrane-bound form (Nagatsu et al. 1964,

Kuczenski & Mandell 1972, Kuhn et al. 1990). In rat

striatal tissue the membrane-bound form seems to

predominate (Kuczenski & Mandell 1972).

It is generally considered that the TH-catalysed

reaction normally is the rate-limiting (committed) step

in the biosynthesis of NA in the PNS and of DA and

NA in the CNS (see also below). In most sympa-

thetically innervated tissues and the brain, the catalytic

activities of DDC and DBH are considered to be high

relative to that of TH. This may be due partly to the

presence of less TH enzyme protein, to a relatively

low catalytic turnover number or to its special regu-

latory properties (see below). Furthermore, the TH

reaction requires an adequate supply of the co-sub-

strate (co-factor) BH4, which depends on three

enzymes for its biosynthesis and one for its contin-

uous regeneration to the catalytically active form (see

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Acta Physiol Scand 2000, 168, 1±17 T Flatmark � Regulation of catecholamine biosynthesis

below and Fig. 2). In addition, four splice variants of

the human enzyme exist (Le Bourdelles et al. 1991),

although the physiological signi®cance of these

isoforms remains to be established. The successful

expression of recombinant hTH isoforms and rTH

and the determination of the crystal structure of the

catalytic domain of rTH (Goodwill et al. 1997, 1998)

have given valuable new information in terms of the

structure and function of this enzyme (reviewed in

Flatmark & Stevens 1999a).

Biosynthesis and regeneration of the co-factor tetrahydrobiopterin

GTP cyclohydrolase 1 (GTPCH 1, EC 3.5.4.16)

catalyses the formation of 7,8-dihydroneopterin

triphosphate from GTP and normally represents the

rate-limiting step in the biosynthesis of the natural

co-factor tetrahydrobiopterin (BH4) of the TH-

catalysed hydroxylation of L-Tyr (Fig. 2). The enzyme,

which is a homodecamer (28-kDa subunit) is expressed

in all CA and serotonin-producing cells, but the level of

basal expression in the rat brain varies with the trans-

mitter speci®city of the neurones. This explains the

clinical symptoms observed in humans with a de®-

ciency in BH4 biosynthesis as a result of mutations in

the GTPCH 1 gene. GTPCH 1 is subject to feedback

inhibition by BH4, mediated by a BH4-dependent

complex formation between a 35-kDa regulatory

protein and GTPCH 1 (Milstien et al. 1996). The two

other enzymes in the pathway (Fig. 2), i.e. 6-pyruvoyl-

tetrahydropterin synthase (PTPS, EC 4.6.1.10) (Taki-

kawa et al. 1986) and sepiapterin reductase (SR, EC

1.1.1.153) (Smith 1987), are normally not rate-limiting

in neuroendocrine cells, but PTPS can be so in enzyme

de®ciencies caused by mutations in the human PTPS

gene.

From Fig. 2 it is also seen that the oxidation of

BH4 in the TH reaction generates a 4a-hydroxy-tetra-

hydrobiopterin (pterin-4a-carbinolamine) intermediate

(Lazarus et al. 1983, Haavik & Flatmark 1987) as well as

a cyclic intermediate (AlmaÊs et al. 1996). These

intermediates are subsequently dehydrated to

quinonoid-dihydrobiopterin (q-BH2) and water (Fig. 2)

by pterin-4a-carbinolamine dehydratase (PCD, EC

4.2.1.-) (Rebrin et al. 1995, KoÈster et al. 1995, AlmaÊs

et al. 1996).

Although not directly involved in CA biosynthesis,

dihydropteridine reductase (DHPR, EC 1.6.99.7) is

intimately linked to the TH-catalysed hydroxylation.

DHPR is a homodimer (26-kDa subunit) and catalyses

the reduction of the quinonoid-dihydropterin that has

been formed during the hydroxylation of L-Tyr (Fig. 2).

As BH4 is a speci®c co-factor (co-substrate), a reduc-

tion in the activity of DHPR would effectively reduce

the availability of BH4 and the activity of TH, and thus

may become rate-limiting as seen in inborn errors of

DHPR de®ciency (see below). As expected, the distri-

bution of this enzyme activity in the brain does not

appear to parallel the CA or the serotonin content of

brain tissue as BH4 participates in other reactions

besides the hydroxylation of L-Tyr and L-tryptophan

(Kaufman et al. 1990, Marletta et al. 1998).

3,4-Dihydroxy-L-phenylalanine (L-DOPA) decarboxylase

Another enzyme involved in CA biosynthesis is

L-DOPA decarboxylase (DDC, EC 4.1.1.28) (Fig. 1),

the ®rst CA biosynthetic enzyme to be discovered

(Blaschko 1939). DDC catalyses the decarboxylation

of not only L-DOPA and its analogues but of

all naturally occurring aromatic L-amino acids as well

as of 3,4-dihydroxy-L-phenylserine (see below) and

5-hydroxy-L-tryptophan, and is therefore also referred

to as L-aromatic amino acid decarboxylase. It is a

homodimeric cytosolic enzyme and the recombinant

kidney enzyme contains one catalytically active pyri-

doxal 5¢-phosphate (vitamin B6) active site per subunit

(Moore et al. 1996). Its high catalytic activity in neuro-

endocrine cells may explain why it has been dif®cult to

detect endogenous L-DOPA in sympathetically inner-

vated tissue and brain. It is rather ubiquitous in nature,

occurring in the cytosplasm of most tissues including

the adrenal medulla, brain, kidney, liver and stomach at

high levels, suggesting that its function in metabolism is

not limited solely to CA biosynthesis.

Dopamine b-hydroxylase

Hagen & Welch (1956) showed that brain, sympathet-

ically innervated tissue, sympathetic ganglia and adrenal

medulla can transform DA into NA by hydroxylation

of DA at the beta carbon, but it was not until 1965 that

the enzyme responsible for this conversion was puri®ed

from bovine adrenal medulla (Friedman & Kaufman

1965). The enzyme, dopamine b-hydroxylase (DBH,

dopamine b-monooxygenase, EC 1.14.17.1), is a mixed

function oxidase (reviewed in Skotland & Ljones 1979)

which requires dioxygen and utilizes ascorbate (AH± )

as the physiological single electron donor (Terland &

Flatmark 1975)(Fig. 3). DBH is a homotetrameric and

highly antigenic glycoprotein of 290 kDa (bovine

enzyme) and requires 1 Cu/subunit for catalytic activity

(Abudu et al. 1998). It is associated with the CA storage

organelles in NA/A synthesizing neuroendocrine cells,

partly as a soluble and partly as a membrane-bound

enzyme (Bjerrum et al. 1979, Skotland & Flatmark

1979). The enzyme has a rather low substrate speci®city

and acts in vitro on a variety of substrates besides DA,

hydroxylating almost any phenylethylamine to its

corresponding phenylethanolamine. Thus, a number of

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Regulation of catecholamine biosynthesis � T Flatmark Acta Physiol Scand 2000, 168, 1±17

the resultant, structurally analogous metabolites can

replace NA at the noradrenergic nerve endings and

function as `false neurotransmitters'. The localization of

the enzyme within the neurotransmitter- or hormone-

containing storage vesicle requires a system for the

regeneration of ascorbate from ascorbate-free radical

(AFR) produced in the hydroxylation reaction (Fig. 3).

Cytochrome b561, an integral transmembrane haeme-

protein (Flatmark & Terland 1971), is essential in the

regeneration of intravesicular ascorbate from AFR by a

transmembrane electron transfer (Njus & Kelly 1993)

in which the extravesicular electron donor probably is

cytosolic ascorbate (Flatmark & Terland 1971, Njus

et al. 1987).

Phenylethanolamine N-methyl transferase

In the adrenal medulla NA is N-methylated by the

enzyme phenylethanolamine N-methyl transferase

(PNMT, EC 2.1.1.28) to form A. This enzyme is largely

restricted to the adrenal medulla (Wong et al. 1987)

although low levels of activity and immunoreactive

protein have been reported in the mammalian brain

(Diaz Borges et al. 1978), especially in areas involved

with olfaction, and in the heart and lung a non-speci®c

N-methyltransferase (NMT) has been reported

(Kennedy et al. 1990). Based on studies in transgenic

mice experimental evidence has been presented that

PNMT is not rate limiting for A synthesis in the CNS

(Cadd et al. 1992). It is a cytosolic enzyme and the

methyl donor S-adenosylmethionine is required for the

methylation reaction. The adrenal medullary enzyme

has a rather low substrate speci®city and transfer

methyl groups to the nitrogen atom on a variety of

beta-hydroxylated amines (beta-phenylethanolamines)

(Grunewald et al. 1988). Recombinant forms of bovine

(Park et al. 1991) and human (Caine et al. 1996) PNMT

have been expressed and partially characterized. The

Figure 1 The catecholamine biosynthetic

pathway. For abbreviations, see Appendix 1.

Slightly modi®ed from Blaschko (1939).

Ó 2000 Scandinavian Physiological Society 5

Acta Physiol Scand 2000, 168, 1±17 T Flatmark � Regulation of catecholamine biosynthesis

Km-values for phenylethanolamine and S-adenosylme-

thionine range from 12 to 46 lM and 1.1 to 1.5 lM,

respectively, for four reported `isoforms' of the bovine

enzyme (Wong et al. 1987).

REGULATION OF CATECHOLAMINE

BIOSYNTHESIS

The CAs in neuroendocrine cells are in a state of

constant ¯ux. They are continuously being synthesized,

released and metabolized, yet they maintain a remark-

able constant level in tissues (reviewed in Axelrod 1971,

von Euler 1971). CA levels and their physiological

actions may thus be regulated at many sites. These

include the ease of their release, the type and sensitivity

of the multiple receptors in target cells, the ef®cacy of

the monoamine reuptake system in catecholaminergic

secretory cells allowing for repeated reuse of the same

molecules (Hertting & Axelrod 1961), as well as a close

regulation of CA biosynthesis (Weiner 1970) and

degradation. In the present review the main emphasis

will be on the recent studies on the long-term and

short-term mechanisms involved in the regulation/

modulation of CA biosynthesis in neuroendocrine cells,

with special reference to physiologically relevant

mechanisms. The enzymes involved seem to be regu-

lated by a variety of physiological factors, both on a

long-term scale (hours to days) as well as the short-term

basis (seconds to minutes), including the relative rates

of synthesis, degradation and state of activation of the

enzymes, notably of tyrosine hydroxylase (TH) which

normally represents the critical control point in the

biosynthesis of all CAs.

Long-term regulation by modulation of TH gene expression

TH is constitutively expressed in a restricted number of

areas in the CNS (catecholaminergic neurones), the

Figure 3 Schematic presentation of the hydroxylation of dopamine

(DA) catalysed by the membrane-bound form of dopamine

b-hydroxylase (DBH) within the secretory vesicle. Abbreviations

additional to main list: ARF, ascorbate free radical A±; AH±, ascorbate

(single electron donor); Cyt b561, cytochrome b561 (mediates trans-

membrane electron transfer for regeneration of ascorbate); H+-

ATPase, vacuolar proton ATPase [generating a proton electrochem-

ical gradient, i.e. a pH gradient (acidic inside) and a membrane

potential (negative inside)]; VMAT, vesicular monoamine transporter

(catalysing an electrogenic H+/monoamine antiport); ± and + indicate

the membrane potential.

Figure 2 Schematic presentation of the

hydroxylation reaction catalysed by cytosolic

and membrane-bound tyrosine hydroxylase.

For abbreviations, see Appendix 1. The rate-

limiting enzymes tyrosine hydroxylase (TH)

and GTP cyclohydrolase 1 (GTPCH 1) in bold-

face.

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Regulation of catecholamine biosynthesis � T Flatmark Acta Physiol Scand 2000, 168, 1±17

sympathetic nervous system and the chromaf®n cells of

adrenal medulla, and independent studies in a large

number of laboratories have revealed that its activity is

under stringent control through both transcriptional

and post-translational mechanisms during develop-

ment/differentiation (Fujinaga & Scott 1997, Lazaroff

et al. 1998) and in adulthood. A growing body of

evidence suggests that different tissues regulate their

TH levels and activities in different ways (Kumer &

Vrana 1996, Liu et al. 1997, Joh et al. 1998). Long-term

regulation of TH occurs by modulation at the level of

TH gene expression (transcription rate and mRNA

stability), and the enzyme appears to be regulated in a

complex modular fashion by both positive and negative

regulatory elements of the TH gene (Liu et al. 1997).

Thus, the expression of the enzyme is elevated in vivo

and in vitro (cell culture) in response to a variety of

trans-synaptic signals, hormones, growth factors and

stressors. Multiple surface receptors and signalling

pathways are involved including three major second

messengers, i.e. cAMP, diacylglycerol and [Ca2+]. Two

transcription factor-binding sites (cis-regulatory ele-

ments) present in the proximal region of the TH gene,

the cAMP-responsive element (CRE) and the TPA-

responsive element (TRE), have been shown to play

important roles in TH regulation both in vitro and in vivo.

Thus, an increase in TH mRNA and activity levels has

been demonstrated in cell cultures by increased level of

cAMP (Tank et al. 1986, Lewis et al. 1987, Melia et al.

1992) and protein kinase C activation (Vyas et al. 1990)

as well as to some extent by glucocorticoids (Baetge

et al. 1981). Additional cis-regulatory elements

governing transcriptional activation of the rat TH gene

have been reported, including an AP-1 domain medi-

ated by the activator protein-1 transcription factor

complex (AP-1 proteins c-Fos, c-Jun and JunD)(Mishra

et al. 1998, Yang & Raizada 1998). Thus, in PC12 cells

the AP-1 activity and TH gene expression increase in

response to, e.g. hypoxia, and the magnitude of the

response depends on the intensity and duration of the

hypoxic stimulus (Mishra et al. 1998). c-Fos was shown

to be essential for functional activation of AP-1.

Furthermore, a physiological reduction in dioxygen

levels induces in the same cells a functional phos-

phorylation of the cAMP-responsive element-binding

protein (CREB) and transcriptional activation of the

TH gene (Beitner-Johnson & Millhorn 1998). Hypoxia

stimulates expression of the c-Fos gene, also reported

to occur in intact animals (Mishra et al. 1998). An

additional effect of hypoxia is an increased stability of

TH mRNA which results from fast enhanced binding

of a cytosolic protein (hypoxia inducible protein) to a

pyrimidine-rich sequence within the 3¢ untranslated

region of TH mRNA (Czyzyk-Krzeska et al. 1997). As

seen from Fig. 2 dioxygen is a substrate in the synthesis

of DA and NA (as well as of serotonin), and changes in

environmental dioxygen appear to cause corresponding

alterations in CA synthesis in neuroendocrine cells

(Davis 1975). Thus, evidence of reduced synthesis of

CA during short-term hypoxia has been reported

(Rostrup 1998). Furthermore, exposure of experimental

animals to alternative stressors (e.g. long-term repeated

immobilization stress) causes an increase in gene

expression and catalytic activity of TH in rat adrenal

medulla (Kvetnansky et al. 1970), in sympathetic ganglia

(Nankova et al. 1996) and in the major central NA

nucleus, locus coeruleus (Rusnak et al. 1998a). Hypo-

glycaemia induced by a single or repeated insulin

administration led to fourfold elevation of adrenal

medullary TH mRNA levels, but not in locus coeruleus

(Rusnak et al. 1998b). Furthermore, hypothalamic NA

is considered to play an important role in the control of

secretion of gonadotropin and sexual behaviour, and

sexual activity in rabbits has been reported to modulate

the expression of TH in locus coeruleus (Yang et al.

1997). Using transgenic mice it has recently been

demonstrated that the CRE and TRE regulatory ele-

ments also play an essential role in the basal expression

of TH in adult tissues in vivo (Trocme et al. 1998).

Induction of TH is observed in vivo under conditions or

with agents that increase the utilization of intracellular

CAs. Examples are cold stress (Baruchin et al. 1990,

Melia et al. 1992), immobilization stress (see above),

and administration of the vesicular amine transport

inhibitor reserpine, which depletes the CA stores

(Faucon Biguet et al. 1991, Hartman et al. 1992).

However, little information is available on the life span

of TH and its contribution to the control of gene

expression, except that hTH has been shown to be a

substrate for the ubiquitin-conjugating enzyme system

(A.P. Dùskeland & T. Flatmark, unpublished data).

Long-term regulation by modulation of bioavailability

of tetrahydrobiopterin (BH4)

TH is considered to be subsaturated with its co-factor

BH4, e.g. based on a comparison of its estimated low

tissue concentrations (<10 lM) in neuroendocrine cells

and the much higher apparent [S]0.5-values observed in

kinetic studies on the TH-catalysed reaction (Flatmark

et al. 1999b). As GTP cyclohydrolase 1 (GTPCH 1) is

the ®rst and rate-limiting enzyme in the de novo synthesis

pathway of BH4 (Fig. 2), a main focus has been set on

this enzyme. Based on quantitative in situ hybridization

(Lentz & Kapatos 1996) and immunocytochemistry

(Dassesse et al. 1997, Hirayama & Kapatos 1998) on rat

brain, evidence has been presented that different cate-

cholaminergic neurones have a highly variable consti-

tutive expression of GTPCH 1 and thereby regulate

their BH4 levels and CA biosynthesis in different ways.

Ó 2000 Scandinavian Physiological Society 7

Acta Physiol Scand 2000, 168, 1±17 T Flatmark � Regulation of catecholamine biosynthesis

From a clinical point of view it is an important

observation that dopamine neurones in the rat brain

contain by far the lowest levels of GTPCH 1 transcripts

(Lentz & Kapatos 1996) and thus appear to synthesize

and maintain BH4 at low levels. This ®nding in rats

offers an explanation for the rather selective dysfunc-

tion of the basal ganglia observed in humans with the

dominant form of L-DOPA responsive dystonia, owing

to mutations in the GTPCH 1 gene (Ichinose et al.

1994, Flatmark & Knappskog 1998). In this disorder a

de®ciency of BH4 within dopaminergic neurones

explains the clinical symptoms from the basal ganglia,

i.e. the affected patients dif®culty in carrying out

voluntary motoric activity. Recent studies in PC12 cells

also support the conclusion that the intracellular BH4

levels are tightly linked to hydroxylation of L-Tyr and

that BH4 bioavailability modulates CA synthesis, regu-

lated by the expression of the gene encoding GTPCH 1

(Serova et al. 1997, Anastasiadis et al. 1998).

Regulation of L-DOPA decarboxylase (DDC ) gene expression

As already discussed above the rather high activity of

DDC in catecholaminergic neuroendocrine cells explain

why it has been dif®cult to detect endogenous L-DOPA

in sympathetically innervated tissue and brain. In spite

of this fact a possible regulatory function of DDC in CA

biosynthesis has been considered (Cumming et al. 1995).

Although no clear answer has been obtained changes in

DDC mRNA, but not in TH mRNA, levels in rat brain

have been observed following long-term antipsychotic

treatment. However, in contrast to the activity of TH

and DBH in the rat superior cervical ganglion that of

DDC is not changed by stimulation of the pre-gangli-

onic cervical sympathetic trunk (Chalazonitis et al.

1980). Similarly, a trans-synaptic induction of TH by

reserpine caused no change in the total activity of DDC

(Hendry 1976). The physiological relevance of these

model studies is, however, uncertain. An interesting

recent observation is the demonstration of human

autoantibodies directed against DDC in patients with

autoimmune polyendocrine syndrome type I (APS I)

(Husebye et al. 1999). This ®nding con®rms and extends

previous observations that APS I patients have inhibi-

tory antibodies against key enzymes involved in neuro-

transmitter biosynthesis.

Regulation of dopamine b-hydroxylase (DBH) gene expression

Dopamine b-hydroxylase (DBH), catalysing the

conversion of DA to NA (Fig. 3), is selectively

expressed in both noradrenergic and adrenergic cells.

Both the membrane-bound and soluble form of DBH

(see above) arise from a single translational product

(Taljanidisz et al. 1989). The expression of the enzyme

is elevated in vivo in response to a variety of trans-

synaptic signals, hormones, growth factors and stress

(reviewed by Sabban & Nankova 1998). In general,

DBH gene expression is activated by a subset of

conditions which elevate TH gene expression both

in vivo and in vitro (tissue culture models). In some

situations they are activated concomitantly, whereas

activation of DBH gene expression often requires

stronger or more prolonged stimuli than of TH

(Sabban & Nankova 1998). Considering the experi-

mental evidence that TH is normally also rate limiting

for NA/A biosynthesis, the physiological signi®cance

of the transcriptional regulation of DBH has yet to be

established.

Regulation of phenylethanolamine N-methyltransferase gene

expression

PNMT is constitutively expressed in A-cells of the

adrenal medulla and in speci®c neurones in the CNS.

From in vitro and in vivo studies performed in several

animal species, including man, it is well documented

that, in the adrenal medulla, PNMT activity is depen-

dent on the high levels of glucocorticoids received from

the cortex (Wurtman & Axelrod 1965, Pohorecky &

Wurtman 1971, Mannelli et al. 1998). Physiological

levels of glucocorticoid hormones are required to

maintain the catalytic activity of PNMT in the rat, and

PNMT gene expression is directly regulated by gluco-

corticoids produced by the adrenal cortex (Stachowiak

et al. 1988, Jiang et al. 1989). A similar glucocorticoid-

dependent regulation has been observed in the superior

cervical ganglia (Stachowiak et al. 1988). On the other

hand, during chronic hypercortisolism, adrenal PNMT

activity is not enhanced, but an overall reduction in

sympatho-adrenal activity is observed (Mannelli et al.

1998). The estimated Kd-value of 1 nM dexamethasone

for the glucocorticoid receptor in chromaf®n cell

primary cultures corresponds to the concentration

required to produce a half-maximal increase in PNMT

activity (Kelner & Pollard 1985). This hormonal action

is speci®cally mediated via a glucocorticoid-responsive

element (GRE) encoded within the 5¢ regulatory region

of the rat PNMT gene (reviewed by Evinger 1998).

Neurally mediated regulation of PNMT expression in

adrenal chromaf®n cells occurs predominantly through

the in¯uence of cholinergic stimuli on nicotinic and

muscarinic receptors. Thus, sequences conveying

responsiveness to nicotinic and muscarinic stimuli also

map to distinct regions of the PNMT promoter

(Evinger 1998). Evidence has been presented that the

gene encoding PNMT is transcriptionally activated by a

synergistic interaction of the glucocorticoid receptor,

the transcription factors AP-2 and Egr-1 (Wong et al.

1998) that undergo translocation to the nucleus. Thus,

8 Ó 2000 Scandinavian Physiological Society

Regulation of catecholamine biosynthesis � T Flatmark Acta Physiol Scand 2000, 168, 1±17

the enzyme is regulated in a different way from TH and

DBH (which are essentially glucocorticoid-indepen-

dent) by its relatively selective modulation by gluco-

corticoids produced in the adrenal cortex (Betito et al.

1994). In addition, cell-speci®c determinants also play

critical roles in specifying the highly restricted expres-

sion of the PNMT gene, e.g. during differentiation

(Evinger 1998).

Post-translational modulation of catecholamine biosynthesis

Among the post-translational mechanisms that have

been shown to be important in the control of TH

activity are phosphorylation, end product (CA) inhibi-

tion and substrate inhibition (reviewed in Kaufman &

Kaufman 1985, Kumer & Vrana 1996). Human, rat and

bovine TH is phosphorylated in vitro by at least seven

protein kinase systems on four serine residues (Ser 8,

19, 31 and 40) (Campbell et al. 1986, Kumer & Vrana

1996). The best characterized of these is phosphoryla-

tion on Ser40 by protein kinase A (PKA), which in vitro

causes an increased af®nity (a reduction in the [S ]0.5-

value) for the co-factor BH4 as well as an increased

negative co-operativity of co-factor binding (Flatmark

et al. 1999b). Furthermore, it also leads to a lower

af®nity for the CA feedback inhibitors, concomitant

with an unchanged or slightly increased Vmax,

depending on the nature of the enzyme preparation

(Kumer & Vrana 1996). Phosphorylation triggers a

conformational change in the protein which also

increases the thermal stability of CA-free recombinant

hTH (MartõÂnez et al. 1996). Other serine/threonine

kinases, like protein kinase C (PKC) and calcium/cal-

modulin-dependent protein kinase II (CaMKII), as well

as MAP kinase-activated protein kinases (MAPKAP

kinases) 1 and 2 are able to phosphorylate hTH1 at

Ser40 to a reported stoichiometry of about one per

subunit (Sutherland et al. 1993). Although an increase in

TH activity after phosphorylation of Ser19 and Ser31

has also been reported in the perfused rat adrenal gland

(Haycock & Wakade 1992), the physiological signi®-

cance of the other phosphorylation sites is not yet clear.

In the perfused gland experiments it was concluded

that the increase in the phosphorylation state in

response to nerve stimulation is mediated by multiple

®rst and second messenger systems which are capable

of signi®cant `cross-talk' (Kumer & Vrana 1996).

However, it is still not known whether the phosphor-

ylation of the various sites is independently regulated

in vivo, or if they have synergistic or antagonistic effects

on each other. Furthermore, the speci®c protein

phosphatase responsible for the modulation of TH

activity by dephosphorylation has not been identi®ed.

High levels of protein phosphatase 1 (PP-1) are present

in the nervous system and protein phosphatase 2A (PP-

2A) has been identi®ed as the major TH phosphatase in

the adrenal medulla and corpus striatum (Haavik et al.

1989). In in vitro studies TH is also subject to a classical

feedback inhibition by all the natural CAs (Kaufman &

Kaufman 1985), which bind with high af®nity to the

active site, competitive with the BH4 co-factor

(Andersson et al. 1988, AlmaÊs et al. 1992). A tight

binding of CAs is visualized by the isolation of TH

from rat and bovine adrenal medulla as enzyme±

Fe(III)±CA complexes with a blue-green colour,

resulting from a charge-transfer transition with

maximum absorbance at 700 nm (Andersson et al.

1988). The physiological signi®cance of this inhibition

is, however, not fully understood. The possibility that

TH is an allosteric enzyme has also been proposed

based on kinetic studies of partially puri®ed rTH from

PC12 cells (Minami et al. 1992) and from corpus stri-

atum (Maruyama & Naoi 1994). For both enzyme

forms an apparent positive co-operativity of co-factor

(BH4) binding as well as very high [S ]0.5-values

(>300 lM) for BH4 was reported (Minami et al. 1992,

Maruyama & Naoi 1994). By contrast, the catechol-

amine-free recombinant hTH, expressed in E. coli,

demonstrates a negative co-operativity of BH4 binding

and a [S]0.5-value of only 24 � 4 lM for BH4, which

matches the estimated cellular concentrations of the

co-factor (Flatmark et al. 1999b). When compared with

data obtained for the recombinant hTH, the kinetic

parameters for the enzyme isolated from biological

materials can be explained by the difference in enzyme

source and nature of the enzyme preparation. Thus, the

TH obtained from cell/tissue extracts is known to yield

a variable amount of enzyme forms containing tightly

bound CAs (Andersson et al. 1988), which bind by

bidentate co-ordination to the active site iron with

displacement of two water molecules (Andersson et al.

1988, Erlandsen et al. 1998) and reversibly inhibit the

enzyme (AlmaÊs et al. 1992). Substrate inhibition (at

[L-Tyr] >50 lM) is regularly observed in vitro and may

play a role in the regulation of CA biosynthesis in vivo

(Kaufman & Kaufman 1985, Quinsey et al. 1996).

Catecholamines and non-shivering thermogenesis

Non-shivering thermogenesis (NST) in response to

cold environment is an example of a physiological

response involving both a short-term and long-term

regulation of the activity of the CA biosynthetic

enzymes. When mammals are exposed to a cold envi-

ronment, the capacity of NST by brown adipose tissue

(BAT) increases in association with the stimulation of

mitochondrial biogenesis and tissue hyperplasia, which

are totally dependent on sympathetic innervation to this

tissue (reviewed in Flatmark & Pedersen 1975). NST is

largely achieved by the uncoupling of oxidative phos-

Ó 2000 Scandinavian Physiological Society 9

Acta Physiol Scand 2000, 168, 1±17 T Flatmark � Regulation of catecholamine biosynthesis

phorylation in BAT mitochondria related to the pres-

ence of a speci®c uncoupling protein (UCP1 or ther-

mogenin) in the mitochondrial inner membrane (Lin &

Klingenberg 1980), dissipating the proton electro-

chemical gradient as heat. The uncoupling protein

UCP1 is expressed exclusively in BAT, and its abun-

dance is regulated by NA and mediated by adrenergic

receptors (Cannon et al. 1996). The probable origin of

the sympathetic nerves in the inter-scapular BAT is the

stellate ganglion (Cannon et al. 1986). Chronic cold

exposure increases TH activity in BAT (Flatmark &

Pedersen 1975) and in adrenal glands (Fregly et al. 1994)

involving a combined transcriptional and post-transla-

tional mechanism of regulation.

NEW INSIGHTS INTO

CATECHOLAMINE FUNCTION

FROM TRANSGENIC MODELS

Recent advances in genetic manipulation of the mouse

have had a major impact on catecholamine research. In

particular, the genetic approach of disrupting the genes

encoding the CA biosynthetic enzymes have been very

informative in de®ning mechanisms by which CAs

exert some of their functions. Thus, unanticipated

phenotypes have been observed at the developmental,

physiological and behavioural levels (reviewed in

Thomas & Palmiter 1998). Some of the transgenic

animals result in rather drastic biological effects, e.g. the

targeted disruption of the TH and DBH genes, which

are relevant to human genetic disorders of TH de®-

ciency and DBH de®ciency (see below). Thus, elimi-

nation of DBH in mice (DBH±/±) is lethal and

indicates that NA/A is essential for fetal survival

(Thomas et al. 1995). A similar phenotype (i.e. perinatal

lethality) is observed on disruption of the TH gene

(Kobayashi et al. 1995, Zhou et al. 1995). Evidence has

been presented that fetal demise is caused by cardio-

vascular failure. Furthermore, the neurotransmitters

play a variety of important roles during nervous system

development. By the rescue of the DBH±/± fetuses by

administration of the synthetic amino acid precursor

3,4-dihydroxy-L-phenylserine, forming NA by decar-

boxylation, adult physiology and behaviour in the

mutant mice have also been studied (Thomas &

Palmiter 1998, Thomas et al. 1998). One example is that

in the DBH±/± mice the normal physiological adap-

tation to cold environment is impaired (see above).

Even at room temperature the DBH±/± mice have a

lower body temperature than do controls and they only

survive 1±2 h at 4 °C, whereas controls survive

`inde®nitely' (Thomas & Palmiter 1998). The explana-

tion for this lack of adaptation is defective function of

the normal non-shivering thermogenesis mediated by

the uncoupling protein (UCP1) in the brown adipose

tissue which is normally controlled by NA (see above).

In this and other tissues NA can be restored to normal

or subnormal levels by substitution therapy with 3,4-

dihydroxy-L-phenylserine (Thomas & Palmiter 1998).

Mice unable to synthesize DA speci®cally in dopa-

minergic neurones have also been created by inacti-

vating the TH gene then restoring TH function in NA

cells by substitution therapy with L-DOPA (Zhou &

Palmiter 1995). These studies have revealed that DA is

essential for movement (as expected) and feeding, but

is not required for the development of neural circuits

that control these behaviours.

During embryogenesis, TH expression appears

permanently within cells destined to be CA-producing/

secreting during adult life, and transiently in several cell

types that normally will not express TH in adulthood

(Hess & Wilson 1991). Transgenic mouse models have

been developed to study trans-synaptic regulation of

TH gene expression. This approach has shown that a

9-kb of rTH 5¢ ¯anking sequence mediates cell type-

speci®c trans-synaptic regulation of reporter gene

expression (Min et al. 1996) whereas a 3.6-kb fragment

was inactive (Morgan et al. 1996). Furthermore, by

mutation analyses of the 5¢ upstream DNA sequence of

rTH attempts have been made to map regulatory ele-

ments directing TH expression in CNS cell lines

(Lazaroff et al. 1995).

Transgenic mice have also given valuable informa-

tion on the complex regulatory mechanisms for hTH

gene expression and for CA levels. Thus, mice carrying

multiple copies of the hTH gene in brain and adrenal

medulla have revealed a 50-fold increase in the TH

mRNA level in speci®c regions of the brain as well as

an increase in immunoreactive TH and TH activity.

However, CA levels in the transgenics were not

signi®cantly different from that of non-transgenic

(Nagatsu 1991).

MUTATIONS IN GENES OF THE

CATECHOLAMINE BIOSYNTHETIC

ENZYMES AND HUMAN DISEASES

The discovery of mutations in almost all the human CA

biosynthetic enzymes shown in Fig. 2 have provided

important clues to the pathophysiology of some

neurogenetic disorders as well as to their diagnosis and

treatment. Furthermore, the mutations have been

complementary to the studies on transgenic animals

and contributed signi®cantly to elucidate basic regula-

tory properties of mammalian CA biosynthesis. Human

mutations have at this point been detected for six of the

enzymes and their related clinical and metabolic

phenotypes described (reviewed in Flatmark &

Knappskog 1998). The ®rst group represents four

enzymes which are involved in the biosynthesis/

10 Ó 2000 Scandinavian Physiological Society

Regulation of catecholamine biosynthesis � T Flatmark Acta Physiol Scand 2000, 168, 1±17

regeneration of the co-factor BH4 (Fig. 2), the ®fth

enzyme is TH and the last is DBH. BH4 de®ciencies

represent a heterogeneous group of diseases owing to

mutations in two of the three enzymes involved in its

biosynthesis (GTPCH 1 and PTPS) or in the two

recycling enzymes (DHPR and PCD) (Fig. 2). 107

mutations have so far been identi®ed in this group of

enzymes, i.e. in the GTPCH 1 gene (53), PTPS gene

(25), DHPR gene (21) and PCD gene (7), and include

missense, nonsense, frame-shift and RNA splice site

mutations (see BIODEF database http://www.u-

nizh.ch/blau/bh4.html). Different clinical and

biochemical phenotypes de®ne and characterize the

variants (see BIODEF database). The primary enzyme

defect, its severity, outcome of the BH4 challenge,

reactivity with protein speci®c antibodies, and

responses to therapy are some of the criteria used to

de®ne the speci®c defects. The `typical' (severe, general)

forms are characterized by their clinical symptoms and

signs as a result of impaired hydroxylation of the aro-

matic amino acids L-phenylalanine, L-Tyr and L-tryp-

tophan. The de®ciency of BH4 reduces the formation

of DA/NA (and serotonin) as shown by the symptoms

from the CNS and measurement of CSF neurotrans-

mitter metabolites, and they require treatment. Hype-

rphenylalaninemia is also frequently observed due to

reduced hydroxylation of L-phenylalanine in the liver.

The inherited de®ciencies of GTPCH 1, PTPS and

DHPR are clinically characterized by severe neurolog-

ical symptoms unresponsive to the classical L-phenyla-

nine-low diet used in the treatment of phenylketonuria.

It should be noted, however, that the normal and

pathological intraneuronal level of BH4 is not exactly

known. The level in the CSF, which is used for diag-

nostic purposes, is in the range of 23±55 nM as

compared with the [S]0.5-value of TH which is three

orders of magnitude higher (Flatmark & Knappskog

1998, Flatmark et al. 1999b).

Autosomal dominantly inherited reduction of

GTPCH 1 activity (Ichinose et al. 1994) is often

correlated with the disease ®rst described by Segawa

(Segawa's syndrome) and characterized by progressive

dystonia with marked diurnal ¯uctuations. Patients

respond to relatively low doses of L-DOPA and this

form of GTPCH 1 de®ciency is therefore termed

L-DOPA responsive dystonia (DRD).

So far, the enzymatic phenotype of two mutations in

the hTH gene have been described and characterized in

patients with an early manifestation of DRD (Q381K

mutation) (Knappskog et al. 1995) and in a patient with

juvenile Parkinsonism (L205P)(LuÈdecke et al. 1996)

with favourable long-term response to L-DOPA. The

DRD patients revealed symptoms similar to that

observed for the dominant form of GTPCH 1 de®-

ciency (Ichinose et al. 1994). The expression analyses of

these mutant hTH proteins have identi®ed the contri-

bution of three molecular mechanisms for the reduced

hydroxylation of L-Tyr in this group of patients

(reviewed in Flatmark & Knappskog 1998), i.e. (i) a

reduced stability of the mutant enzyme when expressed

transiently in eukaryotic cells, (ii) an almost complete

loss of enzyme activity of the isolated recombinant

enzyme, and (iii) a kinetic variant form with reduced

af®nity for the substrates and reduced speci®c activity

of the mutant enzyme at physiologically relevant

substrate concentrations. The structural effects of the

mutations in hTH at codons 205 and 381 have recently

been described based on the crystal structure of a

truncated form of rTH containing the catalytic and

tetramerization domains (Goodwill et al. 1997, Flatmark

& Stevens 1999a).

NA and A are crucial determinants of the minute-to-

minute neural regulation of blood pressure and

congenital DBH de®ciency results in severe orthostatic

hypotension, NA failure, and ptosis of the eyelids

(reviewed in Robertson & Hale 1998). There is virtually

absence of NA, A and their metabolites whereas DA is

greatly increased in plasma, CSF and urine. The DBH-

de®cient patients have DA rather than NA in their

neurones. Although the precise genetic cause of DBH

de®ciency has still not been reported, it is clear that

there is no recognizable DBH enzyme in some of the

patients (Robertson & Hale 1998). As for the DBH±/±

transgenic mice the DBH-de®cient patients can be

successfully treated by substitution therapy with 3,4-

dihydroxy-L-phenylserine which is decarboxylated to

yield NA in NA-ergic neurones. A similar neuro-

chemical pattern is observed in patients with partial

DBH-de®ciency associated with mutations in a copper

transporter gene, ATP7A, encoding a highly conserved

copper-transporting ATPase (Kaler et al. 1998).

Presumably this ATPase of the Golgi network is

normally required to incorporate copper into DBH

apoenzyme during processing in the secretory pathway.

Successful response to early copper replacement ther-

apy has been reported.

CONCLUDING REMARKS

The signi®cant advances which have been made in the

past decade regarding the catecholaminergic systems

have opened new avenues for future research. Thus,

considerable progress has been made in our under-

standing of the transcriptional regulation of genes

encoding CA biosynthetic enzymes, transporters and

receptors, although studies on the regulatory properties

have often been performed on in vitro models (cultured

cells), and their relevance for the in vivo situation is not

always clear. Further studies on the catecholaminergic

systems is likely to apply the DNA microassay (DNA

Ó 2000 Scandinavian Physiological Society 11

Acta Physiol Scand 2000, 168, 1±17 T Flatmark � Regulation of catecholamine biosynthesis

`chip') technology to simultaneously monitor the levels

of a larger number of transcripts. On the basis of such

data it will become possible to group more CA-related

genes together based on similar patterns of expression

and, importantly, that functional relationships can be

predicted from these patterns. One problem in inter-

preting patterns of gene expression in the brain in

catecholaminergic neurones is its cellular complexity,

which requires a method for examining gene expression

at the single cell level. Future research will focus more

on the cell speci®c regulatory mechanism of CA

biosynthetic enzymes. The transgenic technology has

already been documented and seems likely to continue

to have a major impact on almost every branch of

neuroscience, including the catecholaminergic systems.

Furthermore, the genetic approach to the study of CA

function will certainly continue including the abnormal

CA function related to an expected increase in the

number of human genetic disorders. The problems of

extrapolation from in vitro studies to the in vivo situation

is also highly relevant for the transcriptional and post-

translational regulation of CA biosynthetic enzymes,

notably of the normally rate-limiting enzyme TH.

This work was supported by grants from the Research Council of

Norway, the Norwegian Council of Cardiovascular Diseases, the

Norwegian Cancer Society, Rebergs legat, the Novo Nordisk

Foundation and the European Commission.

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APPENDIX 1

Selected abbreviations and acronyms

A adrenaline

BH4 tetrahydrobiopterin [(6R)-L-erythro-5,6,7,8-

tetrahydrobiopterin]

CA catecholamine

CaMKII calcium/calmodulin-dependent protein

kinase II

CNS central nervous system

CRE cAMP-responsive element

CREB cAMP-responsive element-binding

protein

CSF cerebrospinal ¯uid

DA dopamine

DBH dopamine b-hydroxylase (EC 1.14.17.1)

DDC 3,4-dihydroxy-L-phenylalanine

decarboxylase (EC 4.1.1.28)

DHPR dihydropteridine reductase (EC 1.6.99.7)

GC±MS gas chromatography±mass spectrometry

GTPCH 1 GTP cyclohydrolase 1 (EC 3.5.4.16)

HPLC high performance liquid chromatography

L-DOPA 3,4-dihydroxy-L-phenylalanine

L-Phe L-phenylalanine

L-Tyr L-tyrosine

MAPKAP MAP kinase-activated protein kinase

NA noradrenaline

NMT non-speci®c N-methyltransferase

16 Ó 2000 Scandinavian Physiological Society

Regulation of catecholamine biosynthesis � T Flatmark Acta Physiol Scand 2000, 168, 1±17

PCD pterin-4a-carbinolamine dehydratase

(EC4.2.1-)

PET positron emission tomography

PKA protein kinase A (cyclic AMP-dependent +

protein kinase, EC 2.7.1.37)

PKC protein kinase C superfamily (EC 2.7.1.37)

PNMT phenylethylamine N-methyl transferase

(EC 2.1.1.28)

PNS peripheral nervous system

PTPS 6-pyruvoyl-tetrahydropterin synthase (EC

4.6.1.10)

SR sepiapterin reductase (EC 1.1.1.153)

TH tyrosine hydroxylase (EC 1.14.16.2)

TPA 12-O-tetradecanoylphorbol-13-acetate

TRE TPA-responsive element

Ó 2000 Scandinavian Physiological Society 17

Acta Physiol Scand 2000, 168, 1±17 T Flatmark � Regulation of catecholamine biosynthesis