Báo cáo khoa học: Characterization of the serotoninergic system in the C57BL/6 mouse skin potx

Characterization of the serotoninergic system in the C57BL/6mouse skin Andrzej Slominski1, Alexander Pisarchik1, Igor Semak2, Trevor Sweatman3and Jacobo Wortsman4 1 Department of Patholo

Characterization of the serotoninergic system in the C57BL/6

mouse skin

Andrzej Slominski1, Alexander Pisarchik1, Igor Semak2, Trevor Sweatman3and Jacobo Wortsman4 1

Department of Pathology, University of Tennessee Health Science Center, Memphis, TN, USA;2Department of Biochemistry, Belarus State University, Minsk, Belarus;3Pharmacology, University of Tennessee Health Science Center, Memphis, TN, USA;

4

Department of Internal Medicine, Southern Illinois University, Springfield, IL, USA

We showed expression of the tryptophan hydroxylase gene

and of tryptophan hydroxylase protein immunoreactivity in

mouse skin and skin cells Extracts from skin and

melano-cyte samples acetylated serotonin to N-acetylserotonin and

tryptamine to N-acetyltryptamine A different enzyme from

arylalkylamine N-acetyltransferase mediated this reaction,

as this gene was defective in the C57BL6 mouse, coding

predominantly for a protein without enzymatic activity

Serotonin (but not tryptamine) acetylation varied according

to hair cycle phase and anatomic location Serotonin was

also metabolized to 5-hydroxytryptophol and

5-hydroxy-indole acetic acid, probably through stepwise

transform-ation catalyzed by monoamine oxidase, aldehyde

dehydrogenase and aldehyde reductase Activity of the melatonin-forming enzyme hydroxyindole-O-methyltrans-ferase was notably below detectable levels in all samples of mouse corporal skin, although it was detectable at low levels

in the ears and in Cloudman melanoma (derived from the DBA/2 J mouse strain) In conclusion, mouse skin has the molecular and biochemical apparatus necessary to produce and metabolize serotonin and N-acetylserotonin, and its activity is determined by topography, physiological status of the skin, cell type and mouse strain

Keywords: mouse skin; serotonin acetylation; arylalkylamine N-acetyltransferase; tryptophan hydroxylase; hair cycle

The skin is the largest body organ and functions as a

metabolically active biological barrier regulating internal

homeostasis and separating the internal milieu from

noxious environmental factors [1] These functions are

mediated by the skin immune, pigmentary,

neuroendo-crine, adnexal and vascular systems [1–7] Most recently

we have uncovered local serotoninergic and

melatoniner-gic systems as novel elements of the cutaneous

neuro-endocrine components of human and hamster skin [8–12]

Serotonin is the product of a multistep metabolic pathway

that starts with the hydroxylation of the aromatic

aminoacid L-tryptophan by tryptophan hydroxylase

(TPH) [13,14] Serotonin can be acetylated by

arylalkyl-amine N-acetyltransferase (AANAT) to N-acetylserotonin

(NAS), which is further transformed to melatonin by hydroxyindole-O-methyltransferase (HIOMT) [13,15] Serotonin can act as a neurohormone, regulator of vascular tone, immunomodulator and growth factor, while melatonin can act as a hormone, neurotransmitter, cytokine or biological modifier [2,15–17] Some of these functions may be pertinent to skin physiology, which exhibits basic differences among the mammalian species

In rodents (mostly nocturnal animals) the skin is shielded from the damaging effect of solar radiation by fur [18], and the morphology of the entire mouse skin changes in close coordination with the cyclic activity of the hair follicle [19] Mouse hair follicle cycling is characterized by

a precisely regulated, time frame-restricted and differential pattern in the expression and activity of melanogenesis related proteins, PH, pterins and thioredoxin reductase [20] Hair cycle-dependent changes also involve adrenergic innervation and specific patterns of b2-adrenergic receptor expression [21]

Our previous studies raised the possibility that the level

of activity of an endogenous serotoninergic pathway would specifically determine whether its products are for internal use (intracrine regulation), or for external secretion

(para-or autocrine regulation) Because mouse skin differs from human skin, we anticipated interspecies heterogeneity in the cutaneous expression of elements of the serotoninergic pathway Therefore, we have tested the expression of dif-ferent elements of the serotoninergic system in the C57BL/

6 mouse and related their activity in the skin to the phase of the hair cycle and to the cutaneous cellular compartments

Correspondence to A T Slominski, Department of Pathology,

University of Tennessee Health Science Center, 930 Madison Ave

Rm 519, Memphis, TN, USA.

Fax: + 1 901 448 6979, Tel.: + 1 901 448 3741,

E-mail: aslominski@utmem.edu

Abbreviations: TPH, tryptophan hydroxylase; AANAT,

arylalkyl-amine N-acetyltransferase; NAS, N-acetylserotonin; HIOMT,

hydroxyindole-O-methyltransferase; PH, phenylalanine hydroxylase;

CDL, curved desolvation line; TBST, Tris buffered saline with Tween

20; NAT, arylamine N-acetyltransferase; Bis, bisubstrate analog

coenzyme A-S-acetyltryptamine; MAO, monoamine oxidase;

5HIAA, 5-hydroxyindolacetic acid; 5HTPOL, 5-hydroxytryptophol.

Note: a website is available at http://www.utmem.edu/pathology

(Received 30 April 2003, revised 4 June 2003, accepted 9 June 2003)

Experimental procedures

Tissue

Murine samples consisted of skin isolated at telogen and

anagen stages of the hair cycle as described previously, and of

brain, pituitary and spleen tissues [12,19] Experiments

performed in the USA used C57BL/6 strain female mice

(8 weeks old) purchased from Taconic (NY) and housed in

community cages at the animal facilities of the Albany

Medical College, Albany, NY LC/MS assays, performed in

Belarus, also used C57BL/6 mice (males 18 weeks old)

obtained from the Vivarium of the Department of

Bio-testings of the Institute of Bioorganic Chemistry (Belarus

State University, Minsk, Belarus) The animals were housed

in a temperature-controlled room on a 12-h light : 12-h dark

schedule (lights turned on at 06.00 h) with food and water

available ad libitum The animals were killed under

pento-barbital anesthesia and selected organs as well as back skin

were collected following protocols routinely used in our

laboratory, and then stored at)80 C until use [12,19] The

Institutional Animal Care and Use Committee at Albany

Medical College approved the original experimental

proto-col, and a similar protocol for mice was approved at

University of Tennessee Health Science Center Approval for

the experiments performed in Minsk, Belarus was granted by

the Belarus University Animal Care and Use Committee

Cell culture

Tested cell lines comprised mouse Cloudman S91 (sublines

6 and M3) and hamster AbC-1 melanoma cells, and mouse

immortalized normal melanocytes (MelA) Melanoma cells

were grown in Ham’s F10 medium as described previously,

and the media were supplemented with 10% (v/v) fetal

bovine serum and 1% antibiotic/antimycotic mixture

(Gibco) [22] MelA (the gift of D Bennett, St George’s

Hospital, London, UK), was cultured in RPMI 1640 media,

supplemented with 10% (v/v) 200 nM bovine serum

(phorbol-12-myristate-13-acetate), in the presence of 10%

(v/v) CO2 After washing with NaCl/Pi, melanoma cells

were detached using Ca- and Mg-free Tyrode’s solution,

containing 1 mMEDTA, while normal immortalized mouse

melanocytes were trypsinized The cells were centrifuged

then frozen at)80oC, for use in further analyses

Enzymatic assays

Arylakylamine/arylamine N-acetyl transferase

acti-vity N-acetyl transferase activity was measured either by

the method of Thomas et al [23], using a modified

RP-HPLC separation with fluorimetric detection of the

reaction products [24] or by direct LC/MS detection of

metabolic intermediates [8] For both methods, tissue or cell

samples were homogenized in an ice-cold 0.25Mpotassium

phosphate buffer (pH 6.8) containing 1 mMdithiothreitol,

1 mM EGTA and protease inhibitor cocktail (2 lLÆmL)1

homogenization mixture, Sigma) Homogenates were

cen-trifuged at 15 000 g for 10 min at 4C Supernatants were

used to measure serotonin N-acetyl transferase in the

presence of 1 mMserotonin or tryptamine and 0.5 mMof

acetyl coenzyme A in 0.25M potassium phosphate buffer

(pH 6.8) for 1 h or 1.5 h (when indicated) at 37C The enzymatic reaction was stopped by the addition of HClO4 After centrifugation, the supernatant was subjected to HPLC in a system equipped with a Novapak C18 reverse-phase column (100· 5 mm, 4 lm particle size; 60 A˚ pore size) and a fluorometric detector with excitation and emission wavelengths set at 285 and 360 nm, respectively The elution was carried out isocratically at ambient temperature with a flow rate of 1.5 mLÆmin)1 for the mobile phases chosen according to the amine substrate to be used The mobile phase contained 4 mMsodium 1-octane-sulfonate as an ion-pairing agent, 50 mM ammonium formate (pH 4.0) vs methanol (80 : 20, v/v) for serotonin and (75 : 25, v/v) for tryptamine Elution peaks of N-acetylserotonin and N-acetyltryptamine were verified by coelution with the authentic standards The peak areas were quantified in relation to known concentrations of N-acetylserotonin and N-acetyltryptamine standards Back-ground controls consisted of the reaction mixture incubated without substrate or enzyme source

For LC/MS analysis, the final concentrations of acetyl CoA and serotonin in reaction mixtures were 0.5 mMand

5 mM, respectively Aliquots of the final reaction super-natants (see above) were separated on an LCMS-QP8000a (Shimadzu, Japan) through Restec Allure C18 reverse-phase column (150· 4.6 mm; 5 lm particle size; 60 A˚ pore size) The elution was carried out isocratically at a flow rate of 0.3 mLÆmin)1at 30C by mobile phases consisting of 20% (v/v) methanol and 0.1% (v/v) acetic acid The effluent from the HPLC system was routed to the MS electrospray interface used in the positive mode Nitrogen was used as nebulizing gas MS parameters were as follows: nebulizer gas flow rate was 4.5 LÆmin)1; the electrospray voltage was 4.5 kV; CDL heater temperature was 250C Selected ion monitoring mode was applied to detect ions with m/z¼ 219 The LC/MS workstationCLASS-8000 software was used for system control and data acquisition (Shimadzu, Japan) Quantitative determination of N-acetylserotonin was made by comparing the observed peak areas with the peak areas of known concentrations of the NAS standard Hydroxyindole-O-methyl-transferase activity Hydroxy-indole-O-methyl-transferase activity was assayed as des-cribed previously [24] Briefly, tissue homogenates were centrifuged at 15 000 g for 10 min at 4C Supernatants were used to measure enzymatic activity in the presence of 0.5 mMof S-adenosyl-L-methionine and 1 mMof N-acetyl-serotonin in 0.05M sodium phosphate buffer (pH 7.9) After incubation for 1 h at 37C, the enzymatic reaction was stopped by the addition of HClO4 and, after centri-fugation, the supernatants were subjected to HPLC in the system described above for measurement of acetyl trans-ferase activity, with tryptamine as the substrate Compar-ison with the retention times of the authentic standards identified elution peaks for N-acetylserotonin and melato-nin Protein concentration was determined with a dye-binding method using BSA as the standard [24]

Western blot analysis Cultured cells were detached in Tyrode’s solution plus 1 mM EDTA, centrifuged at 200 g for 10 min at 4C and the cell

pellets were then washed with NaCl/Pi and frozen at

)70 C For protein isolation, frozen cell pellets or skin

samples were homogenized with a glass homogenizer in

ice-cold buffer A containing 20 mM Tris, pH 7.5, 5 mM

EDTA, 120 lgÆmL)1leupeptin, 3 lMpepstatin and 3 mM

amino-ethylbenzene sulfonyl fluoride The homogenates

were centrifuged at 16 000 g for 10 min at 4C to remove

cell debris and centrifuged at 100 000 g for 1 h at 4C The

supernatants representing the cytosol fraction were removed

and stored at)80 C for further analysis Separate aliquots

of 5 lL were used for protein determination using the

Micro Protein Kit (Sigma) Fifty micrograms of protein

were separated on a 12% (w/v) SDS polyacrylamide gel,

transferred to immobilion-P poly(vinylidene difluoride)

membrane (Millipore Corp, Bedford, MA, USA) and

nonspecific binding sites were blocked by incubation in

5% (w/v) nonfat powdered milk in TBST (50 mM Tris,

pH 7.5, 150 mMNaCl, 0.01% Tween 20) for 4 h at room

temperature Immunodetection of the TPH or AANAT

proteins was performed after overnight incubation with

polyclonal rabbit anti-human TPH (dilution 1 : 1000, at

4C) as the primary antibody (Chemicon, Temecula, CA)

or with rabbit anti-(rat AANAT25-200) serum (dilution

1 : 10 000, room temperature; gift of D Klein, NIH,

Bethesda, MD, USA) In parallel incubation we used

preimmune rabbit serum at the same dilution as the

corresponding antiserum (gift of D Klein, NIH) The

following day membranes were washed twice in TBST for

10 min Goat anti-rabbit IgG coupled to horseradish

peroxidase was used as a secondary antibody (dilution

1 : 4000, 1 h) (Santa Cruz Biotechnology) Membranes

were washed twice in TBST and once in TBS Bands were

visualized using ECL reagent (Amersham Pharmacia

Bio-tech) according to the manufacturer’s instructions

RNA extraction and cDNA preparation

Tissues were pulverized in liquid nitrogen using a mortar

and then suspended in Trizol (Invitrogen) and the isolation

of RNA followed the manufacture’s protocol The synthesis

of first-strand cDNA was performed using the Superscript

preamplification system (Invitrogen) Total RNA (5 lg per

reaction) was reverse transcribed according to the

manu-facturer’s protocol, using oligo(dT) as the primer

All samples were standardized for analysis by the

amplification of housekeeping gene GAPDH as described

previously [25] Mouse TPH was amplified by a single PCR,

while serotonin AANAT was amplified by nested PCR The

localization of the primers in corresponding genes is

presented in Figs 1A and 3A The reaction mixture

(25 lL) contained 2.5 mMMgCl2, 0.25Mof each dNTP,

0.4 lMof each primer, 75 mMTris/HCl (pH 8.8), 20 mM

(NH4)2SO4, 0.01% Tween 20 and 1.25 U of Taq DNA

polymerase (Promega) The mixture was heated to 94C for

2.5 min and then amplified for 35 or 30 cycles as specified:

94C for 30 s (denaturation), 60 C for 45 s (annealing) and

72C for 1 min (extension)

TPH was amplified by a single PCR using primers P108

(5¢-CTTTCGAGTCTTTCACTGCACTC-3¢) and P109

(5¢-CATTCATGGCACTAGTTATGCTC-3¢) Exons 1–2

of mouse AANAT were amplified by primers P242

(5¢-GCCTGTGCAGTGTCAGTGACTC-3¢) in the first round and primers P244 (5¢-CGTGTTTGAGATTGAGC GTGAAG-3¢) and P245 (5¢-CTTGTCCCAAAGTGAGC CGATG-3¢) in the second round of PCR Primers for the first PCR of exons 3–4 of mouse AANAT were P145 (5¢-ACTTGGATGAGATCCGGCACTTCC-3¢) and P148 (5¢-GGCTGACTGCCCAGGTGGTGAAG-3¢) Primers for the second round were P146 (5¢-GTCCAGAGCTGT CACTGGGC-3¢) and P147 (5¢-AGGACAGAGCCCT TGCCCTGCTG-3¢) Annealing temperature for the ampli-fication of exons 3 and 4 was 67C

Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining according to the standard protocol used in our laboratory [8,24,25] The identified PCR products were excised from the gel and purified by GFX PCR DNA using the gel band purification kit (Amersham-Pharmacia-Biotech) PCR frag-ments were cloned in pGEM-T easy vector system (Pro-mega) and purified by plasmid purification kit (Qiagen) Sequencing was performed in the Molecular Resource Center at the University of Tennessee HSC (Memphis) using an Applied Biosystems 3100 Genetic Analyzer and the BigDyeTMTerminator Kit

Results

Tryptophan hydroxylase expression Using mouse-specific primers for mouse tryptophan hydroxylase (Fig 1A) we subjected RNA from different tissues and cell lines to RT-PCR The amplified fragments

of 530 bp were sequenced and shown to have complete (100%) homology with the corresponding gene fragment Thus, the tryptophan hydroxylase gene was expressed in the brain, pituitary, spleen, Cloudman S91 melanoma cells, MelA immortalized normal melanocytes, and anagen and catagen skin (growing and involutional phases of the hair cycle, respectively) The TPH gene was either absent (two experiments) or present (one experiment) in telogen (resting phase of the hair cycle) (Fig 1B, Table 1)

Western blot analysis using two different antibodies was performed in cytoplasmic extracts from mouse skin, MelA

Fig 1 TPH mRNA expression in murine tissues and skin cell lines (A) Localization of the primers to the TPH coding exons The numbers correspond to protein coding exons revealed after comparison of mRNA (NM-009414) and genomic DNA (B) Expression of a 530 bp TPH transcript in brain (2), anagen IV (3), anagen V (4), middle anagen VI (5), late anagen VI (6), and telogen skin (7), spleen (8), subline 6 of S91 melanoma (9) and subline M3 of S91 melanoma (10) DNA markers are shown in lanes 1 and 11.

immortalized normal melanocytes, S91 (clone 6) mouse

melanoma cells and pig pineal gland control These tests

identified a specific protein of 53–55 kDa precipitated by

anti-TPH Igs (Fig 2; arrow) Additional proteins of both

higher (83–85 kDa) and lower molecular mass were also

detected by the same antibodies (Fig 2)

AANAT gene expression

Using different pairs of specific primers located at exons 1

and 2, and 3 and 4 of the AANAT gene we subjected RNA

from mouse tissue and normal and malignant melanocytes

to RT-PCR amplification (Fig 3A) RT-PCR with primers

located at exons 1 and 2 demonstrated the presence of a

163 bp fragment in all tissues and cells tested; this fragment

showed 100% homology with the corresponding fragment

of mouse AANAT cDNA (Fig 3B) In addition, an

aberrantly spliced isoform of 252 bp was detected in the

brain, pituitary, spleen and M3 subline of S91 melanoma,

but not in the C57BL/6 mouse skin or the MelA

melano-cytes (Fig 3B, Table 1) This isoform had the insertion of

89 bp from an intron leading to a frame shift after the first

exon Translation of this transcript would produce a protein

of 59 amino acids with a molecular mass of 6.5 kDa, devoid

of enzymatic activity Tests performed with primers located

at exons 3 and 4 of the AANAT gene yielded bands of

187 bp, 289 bp and 118 bp (Fig 2A,C, Table 1) The

187 bp band corresponding to the normal AANAT cDNA

was detected in the brain, pituitary, parental subline M3 of

Cloudman S91 melanoma and anagen IV mouse skin It

was not detected in skin at telogen, anagen V, early and late

anagen VI and catagen phases of the hair cycle (Fig 2C,

Table 1) The 289 bp band represented the aberrantly

spliced isoform described previously by Roseboom et al

[26] with the insertion of a 102 bp fragment that produced a

frame shift; translation of this mRNA should generate an

inactive enzyme [26] It was detected as the predominant

AANAT species in brain, pituitary and anagen IV skin of

the C57BL/6 mouse, but only as a minor component in Cloudman S91 melanoma (Fig 2C, Table 1) The 118 bp band was detected only in the spleen, and had a deletion of

69 bp (24 bp from exon 3 and 45 bp from exon 4) but a preserved reading frame Thus, this transcript would produce a protein with a deletion of 23 amino acids and

an apparent molecular mass of 20.4 kDa It is, however, unclear whether this protein posses enzymatic activity As the deleted fragment does not include any of the residues critical for substrate binding or enzymatic activity, it is highly probable that it may be enzymatically active Western blot analysis showed a protein with the expec-ted size for AANAT (24 kDa) immunoprecipitaexpec-ted by

Table 1 Tissue and cell line expression pattern of TPH and AANAT

genes from mouse source Numbers 118 (GenBank Accession Number

AY131261), 163 (GenBank Accession Number AF004108), 252

(GenBank Accession Number AY131262), 187 (GenBank Accession

Number AF004108) and 289 (GenBank Accession Number

AF004111) represent the size of corresponding transcripts (bp)

detec-ted by RT-PCR.

Specimens TPH

AANAT Exons 1 and 2

AANAT Exons 3 and 4 Brain (+) 163, 252 187, 289

Pituitary (+) 163, 252 187, 289

Skin (anagen IV) (+) 163 (–)187, 289

Skin (anagen V) (+) 163 (–)

Skin (middle anagen VI) (+) 163 (–)

Skin (late anagen VI) (+) 163 (–)

Skin (telogen) (–)(–)(+) 163 (–)

Spleen (+) 163 118

Melanoma S91

(subline M3)

(+) 163, 252 187, 289 MelA melanocytes (+) 163 (–)

Fig 2 Detection of TPH immunoreactive proteins in mouse skin and cultured melanocytes and melanoma cells (A) Immunoprecipitation using rabbit anti-TPH Igs: skin at catagen (1), anagen III (2) and anagen V (3) phases of hair growth, MelA melanocytes (4), Cloudman S91 melanoma (5), pig epiphysis (6) The arrow indicates a TPH-like immunoreactivity of 53 kDa (B) Immunoprecipitation using sheep anti-TPH Igs Molecular masses in kDa are indicated on the left; skin

at telogen (1), anagen III (2), catagen (3) phases of hair growth, MelA melanocytes (4) The arrow indicates a TPH-like immunoreactivity of

53 kDa (C) Immunoprecipitation control for B The panel presents the blot incubated with secondary antibody only Explanation of numbers and arrow is as above (B).

anti-(rat AANAT) serum (anti-rAANAT25-200) in control

rat brain, Cloudman S91 and hamster AbC-1 melanomas

but not in the mouse skin (Fig 4) Detection of this

AANAT-like immunoreactivity appeared to represent true

expression, as it was not seen in control membranes

incubated with preimmune serum

Acetylation of serotonin and tryptamine

by skin extracts

Using the RP-HPLC system with fluorimetric detection or

LC/MS we were able to firmly establish that extracts of

C57BL/6 mouse skin and of cultured normal and malignant

melanocytes after addition of acetyl-CoA transformed

serotonin to N-acetylserotonin (Figs 5 and 6, Tables 2

and 3) In contrast, the acetylation of tryptamine by skin

extracts was less efficient (Table 2)

The experiments with LC/MS confirm NAS identity by

showing the appearance of an adduct ion (M + H)+at

m/z¼ 219 with the same retention time as the

correspond-ing NAS standard, e.g m/z¼ 219 (calculated mass ¼

218 Da) at a retention time of 27 min (Fig 6B) Kinetic

analysis of N-acetylase activity showed Kmand Vmaxvalues

of 0.56 mM and 174 pmolÆh)1 for serotonin substrate,

respectively (Fig 7) We also tested the effect of bisubstrate

analog coenzyme A-S-acetyltryptamine (Bis; a specific

inhibitor of arylalkylamine activity) on the enzymatic

Fig 4 AANAT-like immunoreactivity is absent in C57BL/6 mouse skin and present in hamster AbC-1 and mouse S91 Cloudman melanomas Immunoprecipitation using rabbit anti-AANAT Igs (upper panel) Lower panel presents blots incubated with secondary antibody only Markers in kDa are shown on the left; rat brain (1), hamster AbC-1 melanoma (2), mouse S91 Cloudman melanomas (3), C57BL/6 mouse skin at anagen III (4), anagen V (5) and catagen (6) phases of hair growth Arrow indicates AANAT-like immunoreactivity of 24 kDa.

Fig 5 HPLC chromatogram obtained from reaction mixture in which S-91 melanoma cells were used as the enzyme source Experimental incubation with acetyl CoA and serotonin (A) or tryptamine (B) and corresponding control extracts without amine substrate (C and D) N-acetylserotonin or N-acetyltryptamine indicate the elution position

of corresponding standards.

Fig 3 AANAT mRNA expression in murine tissues and cell lines.

Structure of the murine AANAT gene Open boxes represent exons.

Shadowed and black boxes are fragments of coding sequence located

after the frame shift and cryptic exons, respectively Primers are shown

by arrows (B) Detection of 163 and 252 bp AANAT PCR bands

amplified by primers located at exons 1 and 2 DNA ladder (1, 14),

pituitary (2), brain (3), MelA melanocytes (4), M3 subline of S91

mel-anoma (5), anagen IV (6), anagen V (7), early anagen VI (8), middle

anagen VI (9), late anagen VI (10), and telogen (11) skin; #6 subline of

S91 melanoma (12); telogen stain (13) (C) Expression of 187 and

289 bp AANAT transcripts amplified by primers located at exons 3

and 4 DNA ladder (1), brain (2), pituitary (3), M3 subline of S91

mel-anoma (4), anagen IV (5); late anagen VI (6), telogen skin (7), spleen (8).

activity in skin extracts at a concentration of < 1 lM, and

found that it inhibited serotonin N-acetyltransferase activity

by approximately 65%, with minimal additional effects at

concentrations > 1 l (Fig 6C,D)

Serotonin N-acetylase activity was dependent on the phase of the hair cycle being low in telogen skin, increasing during anagen to a peak at late anagen VI and decreasing during catagen (Table 2) The enzymatic activity towards tryptamine did not show clear hair cycle dependence Thus, the ratio between enzymatic activity toward tryptamine and serotonin changed during hair cycling from approximately

4 in telogen and anagen III to 16 and 17 in middle and late anagen VI, being 14 in catagen The same ratio was 1.5 in the ear and 1 in S91 melanoma cells (Table 3) While testing cultured cells, we noted significantly lower enzymatic activity in preparations of frozen cells as compared to fresh cells (data not shown)

Serotonin and NAS metabolism in mouse skin LC/MS analysis of the reaction products of arylalkylamine/ arylamine activity in mouse corporal skin showed two metabolites with retention times of 19 min and 23.5 min, corresponding to 5-hydroxytryptophol (5HTPOL; m/z of

178 [M + H]+, calculated mass¼ 177 Da) and 5-hydroxy-indolacetic acid (5HIAA; m/z of 198 [M + H]+, calcula-ted mass¼ 197 Da) (Fig 8) Accumulation of both compounds was inhibited by the monoamine oxidase inhibitor pargyline (Fig 8)

HIOMT activity was below the level of detectability in corporal back skin at telogen, anagen VI and catagen phases of the hair cycle (not shown) However, the chromatograms of products from the HIOMT assay did show nine additional fluorescent products, apart from the NAS substrate The pattern of expression of these products changed during progression of the hair cycle (Fig 9) RP-HPLC separation of the reaction products of the HIOMT assay from mouse ear and Cloudman S91 mouse melanoma cells showed weak but detectable transformation

of NAS to a species with a retention time identical to melatonin (Fig 10) These results suggest that both Cloud-man melanoma cells (derived from the DBA 12J mouse) and the ears of the C57BL/6 mouse express HIOMT activity, albeit at low levels The same activity, however, is undetectable in the corporal skin of the C57BL/6 mouse

Discussion

The current study demonstrates that mouse skin and skin cells have the molecular and biochemical apparatus neces-sary to produce and metabolize serotonin and N-acetyl-serotonin Activity of this serotoninergic system varied depending on anatomical location, phase of hair cycle and skin cell type

In this study, we show the specific expression of the tryptophan hydroxylase gene in normal and malignant mouse melanocytes, in anagen and catagen skin, and in pituitary and spleen In telogen skin gene expression was low Expression of the TPH gene in skin and in skin-derived normal and malignant melanocytes was accompanied by detection of TPH immunoreactive protein with its expected molecular mass of 53–55 kDa The molecular mass of newly translated TPH is 51 kDa and it increases to 53–60 kDa after post-translational modification [14,27] Variants of higher and lower molecular mass have also been described and are believed to represent products of enzyme turnover

Fig 6 LC/MS analysis of serotonin transformation to NAS by mouse

skin Control reaction mixture (A) contains only substrates and

cofactors without addition of the extract Experimental incubation

enzyme extracts, acetyl CoA and serotonin without (B) or with (C)

1 l M of Bis The arrow identifies m/z ¼ 219 at a retention time of

26 min, corresponding to the NAS product The dose-dependent effect

of Bis on serotonin N-acetyltransferase activity is shown in (D).

[28,29] In mastocytoma cells, ubiquitination of TPH can

generate species of higher molecular mass (80–93 kDa) as

intermediates in a very fast turnover process driven by

proteasomes, that leads to the final degradation of native

TPH to species of lower molecular mass [28,29] Such

turnover process would be consistent with our detection of

TPH immunoreactive species with the expected relative

molecular mass (53–55 kDa) as well as species of both

higher and lower relative molecular mass Thus, the high

molecular mass TPH-like species may represent ubiquiti-nated TPH, whereas the low molecular mass TPH-like species could represent degradation products However, as alternative splicing has already been reported for the TPH gene [30,31], the possibility that part of the diversity in TPH-like immunoreactivity molecular mass may be due to translation of alternatively spliced TPH mRNAs cannot be totally excluded The observed expression of the TPH gene

in spleen and pituitary deserves further study to assess the possible production of serotonin by these organs

Our extensive molecular analyses of AANAT transcripts

in the C57BL/6 mouse demonstrate genetic defects that result in the predominant transcription of aberrant isoforms encoding protein(s) without enzymatic activities (Table 1) Thus, our data support the conclusion of Roseboom et al [26] that this species is a natural knockdown for the AANAT However, because we document separately the capability of C57BL/6 mouse skin to acetylate serotonin and tryptamine, the above reactions are likely to be catalyzed by arylamine N-acetyltransferase (NAT) NAT

is a cytosolic enzyme that acetylates nitrogen or oxygen atoms of aromatic amines, hydrazines and N-hydroxyl-amines, thus playing an important detoxifying role [32–35] There are two isozymic forms of the enzyme, NAT-1 and NAT-2, encoded by different genetic loci Each isoform has

at least 15 different allelic forms Expression of NAT-1 cDNA in mammalian or bacterial cells has demonstrated that the enzyme is capable of acetylating both endogenously derived arylalkylamines and exogenous arylamines NAT-1

is ubiquitously expressed in different tissues including the skin, and expression of the NAT-1 gene has been demon-strated by in situ hybridization in rat skin [36–38] Therefore,

we suggest that serotonin and tryptamine acetylation by skin extracts of the C57BL/6 mouse is probably mediated by one of the allelic forms of NAT-1

Testing for the effect of Bis uncovered a dual action in the cutaneous acetylation process, e.g significant inhibition

at concentrations equal to or below 1 lM, indicative of selectivity towards arylalkylamines [39], and insensitivity to

Table 2 Hair cycle dependent changes in skin serotonin acetyl

trans-ferase activity Values represent means ± SEM of two to three assays.

Mouse skin

Enzyme Activity pmolÆmin)1Æmg protein)1 (Mean ± SEM) Activity Ratio

(serotonin/

tryptamine) Serotonin Tryptamine

Telogen 3.4 ± 0.33 0.91 ± 0.001 4

Anagen III 5.64 ± 0.55 1.57 ± 0.2 4

Anagen IV 10.52 ± 0.27 1.02 ± 0.05 10

Early anagen VI 12.48 ± 1.3 0.78 ± 0.09 16

Late anagen VI 16.75 ± 2.46 1.0 ± 0.03 17

Catagen 12.68 ± 0.16 0.91 ± 0.001 14

Ears 9.65 ± 0.51 6.1 ± 1.02 1.5

Table 3 Enzymatic activity in normal and malignant melanocytes.

Values represent means ± SEM of two to three assays bd, below

detectability; na, not applicable.

Cell line

Enzyme Activity pmolÆmin)1Æmg protein)1 (Mean ± SEM)

Activity Ratio (serotonin/

tryptamine) Serotonin Tryptamine

S91 melanoma (6) 1.3 ± 0.01 1.02 ± 0.09 1

MelA melanocytes 2.2 ± 0.48 bd na

S91 melanoma (M3) 15.32 ± 0.001 18.6 ± 1.09 1

Fig 7 Michaelis–Menten and Lineweaver–Burk (insert) plots of

N-acetyltransferase activity for serotonin in mouse skin.

Fig 8 LC/MS of products of reaction mixture in which skin extract was incubated with acetyl CoA and serotonin 5-Hydroxytryptophol (arrow pointing to m/z ¼ 178 with a retention time of 20.2 min) was identified in the reaction mixture (A) and its accumulation was inhibited by pargyline (B) HIAA (arrow pointing at m/z ¼ 192 with a retention time of 25 min) was identified in the reaction mixture (C) and its accumulation was inhibited by pargyline (D).

Bis > 1 lM(less than 25% decrease in activity), suggestive

of preferential activity towards arylamines [40] These

results indicate that transformation of serotonin to NAS

in mouse skin extract is mediated by enzymatic activities

different from AANAT Nevertheless, in at least the DBA/

2 J mouse strain (S91 melanoma), the reaction could still be

mediated by AANAT Thus, that cell type expressed both

the correct AANAT transcript and the protein with the

expected molecular mass Therefore, we suggest that

serotonin acetylation is an intrinsic property of rodent skin; moreover depending on species [41] or specific strains the reaction can either be mediated by NAT-1 or AANAT, or

by both NAT-1 and AANAT

The C57BL/6 mouse strain has undetectable production

of melatonin in the pineal gland, and very low-to-undetect-able concentrations in plasma [42] This is in agreement with the genetic defect in AANAT that led Roseboom et al [26]

to postulate this mouse species as a natural melatonin

knockdown It must nevertheless be noted that significant production of melatonin has been reported in peripheral organs of the same species, most notably in bone marrow-derived cells [43,44] Our own studies that document the skin capability to produce NAS raise the possibility of alternative AANAT-independent pathways to produce this obligatory precursor to melatonin in peripheral organs While our enzymatic studies excluded corporal skin of the C57BL/6 mouse as a site of melatonin production, we did detect low HIOMT activity in mouse ears and in S91 melanoma cells Thus, we tentatively agree with the notion that mice may produce melatonin at selected extracranial sites [43,44] Serotonin is a potent biological agent, and as such needs tight regulation at the tissue level [16,17], provided by monoamine oxidase (MAO) pathways MAO deaminates serotonin to 5-hydroxyindoleacetaldehyde, which is further oxidized to 5HIAA by aldehyde dehydrogenase or reduced

to 5-HTPOL by aldehyde reductase [13] Indeed, when serotonin was incubated with skin extracts, 5HIAA and 5-HTPOL were readily detected by LCMS, whereas addi-tion of the MAO inhibitor, pargyline, blocked producaddi-tion

of these compounds This indicates that serotonin degrada-tion in the skin includes its oxidative deaminadegrada-tion The

H2O2produced during this reaction may also be used for the oxidation of serotonin and other indoleamines, similar

to the intestinal metabolism of tyramine [45]

NAS metabolism was extensive and hair cycle-dependent

in mouse back skin, producing several as yet unidentified

Fig 9 HPLC chromatograms of products of HIOMT assays in telogen

(A), anagen VI (B) and catagen (C) skin Numbers over peaks with

retention times different from NAS represent unknown products of

NAS changing metabolism through the different phases of the hair

cycle, e.g telogen (A), anagen VI (B) and catagen (C).

Fig 10 HPLC chromatogram shows transformation of N-acetylsero-tonin to melaN-acetylsero-tonin in ear (A and B) and S91melanoma (C and D) extracts Experimental incubation with N-acetylserotonin (A and C) and corresponding control incubation without N-acetylserotonin (B and D) The numbers represent the elution position of standards: 1, melatonin; 2, N-acetylserotonin.

metabolites of the indoleamine As NAS has been shown to

be a substrate for horseradish peroxidase [46] it is possible

that these metabolites could be the products of NAS

oxidation by skin hemoproteins Although NAS oxidative

mechanisms have not been fully elucidated, it is possible that

its metabolites may include kynuramines (N1

-acetyl-N2-formyl-5-methoxykynuramine and

N-acetyl-5-methoxy-kynuramine), similar to the oxidation of melatonin [47,48]

To summarize, mouse skin has both the capability of

producing serotonin and the machinery for its extensive

metabolism Data currently available suggest the model for

the serotoninergic pathway in the C57BL/6 mouse skin that

is presented in Fig 11 It would involve stepwise

transfor-mation of tryptophan to serotonin including action by

tryptophan hydroxylase, serotonin metabolism by NAT-1

to NAS and further processing to unidentified products

(presumably kynuramine derivatives) An alternative

degra-dation pathway would include MAO with the production

of 5HTPOL and 5HIAA as intermediate products This

interpretation is consistent with the work of Schallreuter

et al [49–51] showing cutaneous synthesis of

tetrahydro-biopterin (a necessary cofactor for TPH) and expression of

MAO-A activity, and of Debiec-Rychter et al [36]

demon-strating NAT-1 gene expression in rodent epidermis

In mice, hair growth is a complex, highly synchronized

process regulating physiology and morphology of the

entire skin [19] We now add serotonin acetylation to the

hair cycle phase-dependent skin functions The constancy

of tryptamine acetylation throughout the hair cycle

emphasizes the selectivity of cutaneous NAT activity for

serotonin Such selectivity could have physiological and

pathological significance because serotonin transformation

to NAS would limit serotonin effects in the skin (pro-edema, vasodilatory, pruritogenic and proinflammatory activities) The further metabolism of NAS in hair cycle-dependent fashion implies an additional regulatory func-tion of NAS in skin physiology

In summary, we present the molecular and biochemical characterization of the apparatus producing and metabo-lizing serotonin and N-acetylserotonin in the skin of C57BL/6 mouse We define further some of the factors determining the activity of this apparatus that include anatomical location, phase of hair cycle and skin cell type

Acknowledgements

We thank Dr D Klein from NIH for antibodies against AANAT, Bis inhibitor and constructive criticism, and Dr D Bennett (St George’s Hospital, London, UK) and Dr V Hearing (NIH) for immortalized mouse melanocytes (MelA) The work was supported in part by grants from the Center of Excellence for Diseases of Connective Tissue, UTHSC, and from the Center of Genomics and Bioinformatics, UTHSC, to AS.

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