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* Clinical Neurocardiology and
Neural Development, National Institute of Neurological Disorders and Stroke and
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; and
Departments of Neuroscience, Anatomy, and Cell Biology, Tufts University Medical School, Boston, Massachusetts, USA
1Correspondence: Building 10, Room 6N252, National Institutes of Health, 10 Center Dr. MSC 1620, Bethesda, MD 20892-1620, USA. E-mail: ge{at}box-g.nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: L-dopa • tyrosine hydroxylase • sympathetic nervous system • melanocyte • melanin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The importance of catecholamine biosynthesis for embryonic development and postnatal survival is illustrated by the mid-gestational lethality in mice lacking TH (2
, 3)
. As expected, tissue levels of norepinephrine and epinephrine in TH null fetuses are severely reduced to <0.5% those of wild-type fetuses (2)
. However, tissue levels of dopamine (DA) in bodies of TH null fetuses are only reduced to 42% the levels of wild-type fetuses. Kobayashi and colleagues (2)
postulated from these observations, "the existence of an alternative mechanism for producing catecholamines, predominantly DA, one that is not mediated by the TH reaction."
A possible alternative mechanism for synthesis of catecholamines involves tyrosinase, the enzyme responsible for the first step in melanin formation (4
, 5)
. In this step, tyrosinase catalyzes the oxidation of tyrosine to dopaquinone (Fig. 1
). L-dopa is produced from dopaquinone during the first of a series of redox reactions leading to the formation of melanin. L-dopa further drives the reaction by functioning as an alternative substrate for tyrosinase and as a cofactor that stimulates catalytic efficiency. In contrast to lack of TH, lack of functional tyrosinase is not lethal and is commonly encountered as oculocutaneous albinism (6)
.
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The possibility that the L-dopa produced by tyrosinase might serve as a precursor for synthesis of catecholamines was suggested by findings that the presence of tyrosinase in pigmented TH null mice partially restores tissue levels of catecholamines above the negligible levels observed in albino mice lacking TH and functional tyrosinase (3)
. These conditions represented unnatural circumstances, however, and an influence of tyrosinase on catecholamine synthesis in normal animals was not demonstrated.
In the present study we examined whether tyrosinase normally contributes to catecholamine synthesis. We focused our attention on DA because of the observations by Kobayashi et al. (2)
that levels of DA are less affected than other catecholamines by lack of TH. Other findings that most DA is produced peripherally and outside of sympathetic nerves (7
8
9
10)
led us to look for a TH-independent source of DA in peripheral tissues.
Our search for a tyrosinase-dependent and TH-independent source of peripheral DA took advantage of two types of TH null mice characterized by the presence or absence of functional tyrosinase (3)
. We examined age-related changes in DA production using C57BL/6J pigmented and albino mice that produced TH and were genetically identical, except for a spontaneously occurring point mutation of the tyrosinase gene (11
, 12)
. The mechanism by which tyrosinase contributes to age-related changes in peripheral DA production was assessed after immunohistochemical staining for tyrosinase and L-aromatic amino acid decarboxylase (L-AADC), the enzyme that converts L-dopa to DA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These mice were pigmented (i.e., tyrosinase was functional). Tyrosinase-deficient mice (i.e., albino animals) that had the TH mutation were created by back crossing heterozygous pigmented TH null carriers onto albino (homozygous tyrosinase C locus-deficient) ICR mice for three generations, as described previously (3)
. Pigmented or albino mice that were heterozygous for the TH mutation were crossed to produce TH null, heterozygous, and wild-type pigmented and albino mice.
Survival of albino and pigmented TH null embryos was facilitated, according to a described method (13)
by providing pregnant breeding dams with 1.0 mg/mL L-dopa (Sigma, St. Louis, MO) in drinking water after E 8.5 until birth. The L-dopa-supplemented drinking water contained 0.25% ascorbic acid, was changed daily, and was shielded from light to minimize auto-oxidation of L-dopa. Supplementation with L-dopa was discontinued at birth. Samples of tissue, plasma, and urine were harvested from 15-day-old pups. Genotypes of TH null, heterozygous, and wild-type animals were determined by PCR analysis of DNA obtained from tail fragments.
The influence of age on tyrosinase-dependent production of catechols was examined using C57BL/6J mice that were genetically identical except for a spontaneously occurring point mutation of the tyrosinase gene (11
, 12)
. Mice with 2 copies of the mutated tyrosinase gene (C57BL/6J-Tyrc-2J, Jackson Laboratory, Bar Harbor, ME, USA) were albino, whereas heterozygotes and homozygous wild-types were pigmented. In contrast to other mutations of the tyrosinase gene where tyrosinase may be present but is not functional, the mutation present in C57BL/6J-Tyrc-2J mice results in an absence of the enzyme (12)
. These animals were studied at 7, 15, 28, and 60 days of age.
The process by which tyrosinase contributes to age-related changes in peripheral DA production was examined by immunohistochemical staining for tyrosinase and L-aromatic amino acid decarboxylase (L-AADC) in skin of 3-, 6-, 9-, and 30-day-old pigmented C57BL/6J mice.
All procedures were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke at the National Institutes of Health.
Sample collections for catechol measurements
Samples of plasma, urine, and tissues were collected from 4 to 13 mice in each of the groups of albino and pigmented TH null, heterozygous, and wild-type animals studied at 15 days of age and from each of the groups of albino and pigmented C57BL/6J mice studied at 7, 15, 28, and 60 days of age. Samples of blood (150-300 µL) were drawn from ether-anesthetized animals by cardiac puncture using heparinized 1 mL syringes equipped with 28.5 or 31 gauge needles. Blood samples were centrifuged at high speed for 1 min in a microcentrifuge and plasma was separated and frozen on dry ice. Mice were killed by decapitation and samples of urine were collected and frozen on dry ice. Whole brain, heart (ventricles), superior cervical ganglia, spleen, stomach (washed in 0.9% saline), duodenum, pancreas, adrenal glands, and kidneys (kidneys collected only in C57BL/6J-Tyrc-2J and C57BL/6J-Tyr++ mice) were harvested and immediately frozen on dry ice. All samples were stored at -70°C before assays of catechols.
Catechol assays
Tissues were weighed (except for adrenals and superior cervical ganglia) and homogenized in a minimum of 4 volumes of ice-cold 0.4 M perchloric acid containing 0.5 mM EDTA. Tissue homogenates were centrifuged at 4°C to separate the supernatants, which were stored at -70°C until assayed for catecholamines. Catechols, including norepinephrine, L-dopa, epinephrine, and DA, were extracted from samples of plasma, urine, and tissue supernatants using an alumina extraction procedure, then separated and quantified by liquid chromatography with electrochemical detection as described previously (14)
.
Immunohistochemical staining
Immunohistochemical staining was performed on 4% paraformaldehyde-fixed, paraffin-embedded 3 µ sections of mouse dorsal skin using an avidin-biotin immunoperoxidase technique. Histological sections were deparaffinized and rehydrated in two changes of xylene and an ethanol series, and endogenous peroxidase activity was quenched with 0.3% H2O2 in anhydrous methanol for 20 min. Sections were then incubated with an antibody recognizing tyrosinase (
PEP7 at a 1:7000 dilution) (15)
or one recognizing L-AADC (from Protos Biotech Corp., New York, NY, USA; 1:100 dilution) in PBS at 4°C overnight. Biotinylated anti-rabbit IgG (Vector, Burlingame, CA, USA) was used at a dilution of 1:500 in PBS containing 2% normal goat serum at 23°C for 30 min. After 30 min incubation with avidin-biotinylated horseradish peroxidase complex (VectaStain ABC Reagent, Vector) in PBS, slides were incubated for 20 min at 23°C with a peroxidase substrate AEC (3-amino-9-ethylcarbazole) solution (Vector), which generates a red to brownish-red color. Sections were counterstained with hematoxylin and mounted in Immu-Mount (Shandon, Pittsburgh, PA, USA).
Melanin was stained using the Fontana-Masson silver stain and slides were stained using hematoxylin and eosin for routine screening.
Statistics
Results for tissue, plasma, and urine levels of catechols are expressed as means ± SE. Differences among groups of animals were determined by ANOVA. Post hoc tests, using Sheffe’s method, were carried out when comparisons involved multiple groups of animals (e.g., pigmented and albino groups of TH wild-type and null animals or albino and pigmented groups of 7-, 15-, 28-, and 60-day-old animals).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In agreement with our previous findings (3)
, the severity of decreases in tissue catecholamines in TH null animals was slightly, but significantly (P<0.003), greater in albino than in pigmented mice.
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In contrast to the >90% reductions of catecholamines in brain, adrenals, and sympathetic ganglia, the consequences of the absence of TH on catecholamines in other tissues (e.g., heart, stomach, skin), plasma, and urine were highly variable depending on the catecholamine produced and whether the mice were albino or pigmented (Fig. 3
).
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Similar to the findings in brain, adrenals, and sympathetic ganglia (Fig. 2)
, the absence of TH resulted in 74% to 99% decreases (P<0.001) in concentrations of norepinephrine in all other tissues examined and 55% to 99% decreases (P<0.001) of norepinephrine concentrations in plasma and urine of albino and pigmented animals (Fig. 3A, B
). Concentrations of norepinephrine in heart, stomach, skin, plasma, and urine again were less severely decreased by absence of TH in pigmented than in albino animals (P<0.001).
In contrast to the consistent decreases in norepinephrine associated with absence of TH, concentrations of DA in peripheral tissues (e.g., heart, stomach, skin) and urine were decreased (P<0.001) by lack of TH only in albino mice (Fig. 3C, D
). Concentrations of DA in peripheral tissues, plasma, and urine of pigmented animals were 3.5- to 48-fold higher (P<0.001) than DA concentrations in albino animals and were not decreased by the absence of TH. In fact, concentrations of DA paradoxically tended to be higher in pigmented TH null animals than in pigmented TH wild-type animals. Brain, adrenals, and sympathetic ganglia were the only tissues examined where lack of TH resulted in decreased (P<0.001) tissue DA concentrations in albino and pigmented animals and where concentrations of DA did not differ among albino and pigmented TH wild-type animals (Fig. 2)
.
Concentrations of L-dopa in peripheral tissues, plasma, and urine remained generally unaffected by lack of TH in albino and pigmented animals (Fig. 3E, F
). In contrast, concentrations of L-dopa were two- to sevenfold higher (P<0.04) in heart, plasma, and urine of pigmented than albino animals. Concentrations of L-dopa were particularly high in the skin of pigmented animals, where they exceeded (P<0.001) concentrations in albino animals by 80- to 130-fold. Concentrations of L-dopa in skin of pigmented mice were many times higher than L-dopa, norepinephrine, and DA concentrations in all other tissues examined. Concentrations of DA were highest in brain (Fig. 2A
), but relative to those of norepinephrine were particularly high in skin and stomach of TH null and wild-type pigmented animals (Fig. 3C
).
Age-dependent influence of tyrosinase on DA production in TH wild-type mice
We examined the influence of age on tyrosinase-dependent DA production using mice that were genetically identical except for a point mutation of the tyrosinase gene. Mice homozygous for the mutation (C57BL/6J-Tyrc-2J) were albino, whereas their heterozygous or homozygous wild-type littermates were pigmented. The results confirmed a tyrosinase-dependent source of peripheral DA, but showed that this source was restricted to the first 2 wk of postnatal development.
Tissue concentrations of DA and norepinephrine showed striking and variable age-dependent changes, depending on the tissue and on whether mice were albino or pigmented (Fig. 4
). In brain, there were parallel age-dependent increases (P<0.001) in tissue concentrations of DA and norepinephrine, with no differences between albino and pigmented animals (Fig. 4A, B
). In peripheral tissues, there were age-related increases (P<0.001) in norepinephrine concentrations in heart and spleen and decreases (P<0.001) in norepinephrine levels in stomach and pancreas, again with little difference among albino and pigmented animals (Fig. 4D, F, H, J
).
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In contrast to the lack of influence of tyrosinase on developmental changes in norepinephrine, there were striking age-dependent influences of tyrosinase on concentrations of DA in most of the peripheral tissues examined (Fig. 4C, E, G, I
). Tissue concentrations of DA in heart, spleen, stomach, and pancreas of 7- and 15-day-old pigmented animals were 2.6- to 9.5-fold higher (P<0.05) than those of albino animals, but decreased to levels similar to albino animals by 28 days of age and did not differ from then on.
The largest differences in tissue concentrations of catecholamines among albino and pigmented animals were observed in skin for L-dopa and DA (Fig. 5
A, B). In 7-day-old mice, levels of L-dopa in skin were 43-fold higher (P<0.001) in pigmented animals than in albino animals (Fig. 5A
). However, in contrast to the age-associated changes in tissue DA, which rapidly decreased after day 7, L-dopa levels in skin of pigmented animals remained elevated (P<0.001) well above levels in albino animals at all ages.
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Tissue concentrations of DA in skin of 7-day-old pigmented mice were particularly high, exceeding (P<0.001) those in skin of albino mice by 113-fold (Fig. 5B
), and even exceeding (P<0.001) by twofold levels of DA in brain (1.07±0.012 vs. 0.51±0.03 nmol/g). DA concentrations in skin of pigmented mice fell precipitously between 7 and 15 days of age, when they were 27-fold higher (P<0.001) in pigmented than in albino skin. After 28 days of age, concentrations of DA in skin did not differ between albino and pigmented animals.
Plasma levels of L-dopa and DA in pigmented and albino animals (Fig. 5C, D
) showed parallel changes with age to changes in DA concentrations, but not L-dopa concentrations in skin. In 7-day-old pigmented mice, plasma L-dopa concentrations were 16-fold higher (P<0.001) and plasma DA concentrations 4.5-fold higher (P<0.002) than concentrations in 7-day-old albino mice. At 15 days of age, plasma concentrations of L-dopa and DA decreased in pigmented animals toward levels observed in albino animals and did not differ from concentrations in albino mice from then on.
Developmental changes in cutaneous tyrosinase and melanin synthesis
Concentrations of DA in skin and other tissues and concentrations of L-dopa and DA in plasma of pigmented animals showed age-associated changes that corresponded with age-associated changes in the distribution of cutaneous tyrosinase in relationship to sites of melanin synthesis (Fig. 6
).
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Melanocytes in the dorsal skin of 3-day-old mice were found at the epidermal:dermal border and deep in the dermis around hair follicles (Fig. 6)
. Tyrosinase was readily detected in the epidermal layers, even though no melanin was being produced at these locations. In contrast, dermal melanocytes were positive for tyrosinase and melanin was visible at these locations with and without Fontana-Masson staining. Beyond the third postnatal day, epidermal melanocytes decreased in number and staining for tyrosinase decreased; by day 15, virtually no tyrosinase was detected in the epidermis. In contrast, follicular melanocytes became progressively more melanized as mice aged. In contrast to the age-related changes in the distribution of tyrosinase, L-AADC was diffusely present in epidermal and dermal layers at all ages.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Among the different tissues examined, there was considerable variation in amounts of DA that depended on tyrosinase. In part, this reflected the local importance of neuronal or chromaffin cell catecholamine production. Thus, in brain, adrenals, and sympathetic ganglia, tyrosinase-dependent production of DA was only evident in TH null animals, indicating that TH was the principal enzyme responsible for DA production in those tissues. In contrast, tyrosinase and not TH was the major determinant of DA levels in all other tissues examined; the extent of this contribution varied widely but was confined to animals up to 15 days of age.
A surprising finding of the present study was that levels of DA in peripheral tissues, urine, and plasma, and plasma levels of L-dopa tended to be higher in pigmented TH null mice than in pigmented TH wild-type animals. Age-related decreases in the above levels and developmental effects of the TH deficiency state may explain this paradoxical finding. The TH null mice were smaller and appeared to be developmentally younger than their wild-type littermates. Probably as a result of these differences, the pigmented TH null animals had peripheral levels of DA and plasma levels of L-dopa that more closely matched the higher levels found in younger animals.
Tyrosinase is a multifunctional enzyme that catalyzes several steps in melanin production, the first and crucial step involving the hydroxylation of tyrosine (5
, 16)
. Although it is well established that L-dopa is produced during melanin synthesis, it is unlikely that tyrosinase is directly responsible for production of DA from L-dopa. A more reasonable explanation, supported by the present neurochemical and immunohistochemical data, is that tyrosinase-dependent DA production requires the additional actions of L-AADC to convert L-dopa to DA. Alternatively, since tyrosine is also a substrate for L-AADC (17)
, it is possible that tyrosine may first be decarboxylated to tyramine, then converted to dopamine by tyrosinase. Tyramine is a substrate for mushroom tyrosinase (18)
, but whether the mammalian enzyme can produce dopamine from tyramine is unclear.
Sources of tyrosinase-dependent DA production
Where is the tyrosinase-dependent L-dopa and dopamine produced? Since tyrosinase is expressed mainly in melanin-producing cells of skin and eye (16)
, these tissues appear to be the most likely source of the tyrosinase-dependent L-dopa and DA. However, during embryogenesis tyrosinase gene expression has been detected in other cells derived from the neural crest, indicating that tyrosinase may not be restricted to differentiated pigment cells (19)
. Tyrosinase can be expressed in neuroblastomas (20
, 21)
, tumors of early childhood derived from neural crest and characterized by excessive production of DA and L-dopa. More specifically, neuroblastoma cells are capable of differentiating into two cell types: melanocyte-like cells that express tyrosinase and produce melanin and neuronal-like cells that express TH and produce L-dopa and DA (20
, 22)
. Although expression of tyrosinase has been detected in certain brain regions of mice (23
, 24)
and in some extracutaneous human tissues (25
, 26)
, overall there is little evidence for the presence of functional tyrosinase in normal peripheral tissues other than skin, hair bulbs, and eye.
With the above considerations in mind and the present data at hand, it seems likely that the high peripheral levels of DA during the first 2 postnatal wk in pigmented mice are derived largely or exclusively from tyrosinase-dependent production of L-dopa in epidermal melanocytes. Levels of L-dopa in skin of pigmented animals were many times higher than levels of catecholamines in most other tissues. Levels of DA in skin of 7-day-old pigmented animals were particularly high, exceeding DA concentrations in whole brain by twofold. Whereas the levels of L-dopa in skin of pigmented animals remained high, those of L-dopa in plasma and of DA in skin fell precipitously during the second week of postnatal development. Thus, as animals develop, the L-dopa produced by tyrosinase in skin is no longer available as a source of plasma L-dopa or as a precursor for DA.
The possibility that the age-associated decrease in availability of cutaneous L-dopa for synthesis of DA reflects an increase in tyrosinase-dependent production of pigment is supported by our immunohistochemical findings of changes in the cutaneous distribution of tyrosinase. During early postnatal development, tyrosinase is present in upper epidermal layers but does not appear to be associated with melanin synthesis. At this stage of development, L-AADC appears to function in sequence with tyrosinase to produce DA. With further development, tyrosinase becomes localized more prominently around the shafts of hair follicles where the enzyme is increasingly associated with melanin formation. The above observations are consistent with a developmental shift in tyrosinase-dependent production of DA to production of melanin.
Functions of tyrosinase-dependent DA production
What is the function of tyrosinase-dependent DA production during early development? Because lack of functional tyrosinase in albinism has little effect on survival, a critical physiological function of tyrosinase-dependent DA production appears unlikely. Nevertheless, other functions are possible. A contribution to sweat gland development is supported by findings that C57BL/6J-Tyrc-2J albino mice exhibit delayed development of sweat glands compared with their pigmented littermates (27)
.
Visual and auditory processing abnormalities are established features of oculocutaneous albinism (6
, 28)
. Higher tissue levels of DA have been found in sensory structures and brain regions involved in visual-spatial and auditory signal processing of pigmented than of albino rats (29
, 30)
. Thus, tyrosinase-dependent production of DA could be involved in the development of neural projections related to sensory processing. The possibility that retinal abnormalities in albinism are related to local tyrosinase-dependent production of L-dopa or DA is supported by findings that addition of L-dopa to albino eyes normalizes patterns of retinal cell development (31)
.
Greater resistance of pigmented than of albino mice and rabbits to the central nervous system DA-depleting actions of dopaminergic neurotoxins (32
33
34)
and the lower reported incidence of Parkinson’s disease in more heavily pigmented than lightly pigmented races (35
36
37)
suggest other possible influences of tyrosinase-dependent DA production in neurodegenerative disorders. The substantial tyrosinase-dependent production of DA also supports a possible therapeutic use of tyrosinase in the treatment of Parkinson’s disease (38)
.
In summary, the present study establishes a previously unrecognized function of tyrosinase as a developmentally specific major determinant of peripheral DA production. This function may lead to improved understanding of physiological and pathological processes involving tyrosinase and dopaminergic neuronal or paracrine systems.
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ACKNOWLEDGMENTS |
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Received for publication January 23, 2003. Accepted for publication March 19, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  L. Zecca, C. Bellei, P. Costi, A. Albertini, E. Monzani, L. Casella, M. Gallorini, L. Bergamaschi, A. Moscatelli, N. J. Turro, et al. New melanic pigments in the human brain that accumulate in aging and block environmental toxic metals PNAS, November 11, 2008; 105(45): 17567 - 17572. [Abstract] [Full Text] [PDF]
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