(Received for publication, December 29, 1995; and in revised form, February 27, 1996)
From the
Biotransformation of [H]serotonin by
cultured hamster skin to
H-metabolites corresponding to N-acetylserotonin (NAS), melatonin, and 5-methoxytryptamine
(5-MT) was demonstrated. This process was time-dependent, with the
highest production of radioactive NAS and melatonin metabolites after 3
and 5 h of incubation followed by a decrease in the rate of metabolite
release into the media. Conversely, the formation of radioactive
metabolite corresponding to 5-MT increased gradually during skin
culture, reaching the highest level after 24 h of incubation. The
production of
H-metabolites, corresponding to NAS,
melatonin, and 5-MT, was stimulated by forskolin with a maximum effect
of forskolin at 10 µM concentration. The gas
chromatographic/mass spectroscopy analysis of the fraction eluting at
the retention time of NAS standard material showed that it contained
NAS, further confirming production and release of NAS into the media by
hamster skin. Therefore, we conclude that mammalian skin can acetylate
serotonin to NAS and postulate that the NAS is further metabolized by
the skin to form melatonin which is subsequently transformed to 5-MT.
Melatonin serves as the main signal molecule which links the photoperiod to metabolic, endocrine, and immunological changes and which is mainly synthesized in the pineal gland, retina, brain, and Harderian gland(1) . Depending of the site of production and target organ it can act as a hormone, neurotransmitter, cytokine, and biological modifier(2) .
Melatonin is a product of a
two-step conversion of serotonin, which involves the acetylation of
serotonin (3) and subsequent methylation by
hydroxyindole-O-methyltransferase(4) . The majority of
melatonin released into the bloodstream is metabolized in the liver and
kidneys(5, 6) , mainly by 6-hydroxylation and
conjugation to glucuronate or sulfate(5, 6) , and to a
minor degree by deacetylation to 5-methoxytryptamine (5-MT), ()which is further
deaminated(5, 6, 7) . In contrast, melatonin
bioconversion at the organ site of synthesis appears to be different
from the metabolism of circulating
melatonin(8, 9, 10) . For example, in the
retina melatonin is first deacetylated to 5-MT, which can then be
deaminated, producing 5-methoxyindoleacetic acid and
5-methoxytryptophol(8, 9) . Melatonin deacetylation to
5-MT was also detected in retinal pigment epithelium and non-mammalian
skin that are target sites for melatonin
bioregulation(8, 9, 10) . Extracranial sites
of both synthesis and metabolism of melatonin have also been
demonstrated in the peripheral blood mononuclear
leukocytes(11) , and according to some authors in the
gastrointestinal tract(12) .
Melatonin production has not previously been demonstrated in skin, which is the largest body organ that can react to external and internal stimuli via the skin immune system(13) , the pigmentary system(14) , and the skin endocrine system(15, 16) . In lower verterbrates skin is a recognized target for melatonin action, e.g. melatonin has lightening activity on the skin(17) . In mammals, it has been reported that melatonin can regulate hair growth(18) , inhibit follicular melanogenesis(19, 20) , and affect proliferation of epidermal keratinocytes (20) and malignant melanocytes(21) . Specific binding sites for melatonin were also detected in the mammalian skin(20) . In addition, we have identified two isozymic forms of arylamine N-acetyltransferase, NAT-1 and NAT-2, in hamster skin of which NAT-2 catalyzed the acetylation of dopamine to N-acetyldopamine and serotonin to NAS, a direct precursor of melatonin(22) . This information has formed the basis for the present studies on the synthesis and degradation of melatonin by mammalian skin.
Preparation of HPLC fractions for GC/MS analysis was performed by initial drying of fraction samples. The dry residue was reconstituted with 1 ml of methanol, vortex mixed for 30 s, and transferred to a glass conical reaction vial. The samples were then evaporated to dryness and reconstituted with 25 µl of ethyl acetate and vortex mixed. For analysis of 5-MT and melatonin, 1 µl of an ethyl acetate reconstituted sample was used for analysis by GC/MS. For analysis of serotonin, NAS, and an unidentified peak, precolumn derivatization was performed by addition of N-methyl-trimethylsilyltrifluoroacetamide (Regis Chemical Co., Morton Grove, IL) to the ethyl acetate reconstituted sample and incubated at 90 °C for 20 min, followed by GC/MS analysis.
For GC/MS identification of NAS in skin culture, biopsies were incubated for 12-14 h in the presence of 10 µM unlabeled serotonin. The media were extracted with chloroform, and the aqueous phase was analyzed by RP-HPLC as described above. Selected HPLC fractions were then analyzed by GC/MS using selective ion monitoring technique.
Figure 1: Time-dependent DNA synthesis in hamster skin cultured in vitro.
After
metabolic labeling with [H]serotonin, the media
and skin biopsies were prepared separately using chloroform extraction.
Both chloroform and aqueous fractions were analyzed by RP-HPLC in the
presence of nonradioactive serotonin, NAT, 5-MT, and melatonin
standards. Fig. 2shows representative elution times of
tritriated serotonin metabolites released into the media after 5 h
incubation in the presence of 5 µCi of
[
H]serotonin. The radioactive peaks with
retention times corresponding to those of unlabeled standards of NAS
(28 min), 5-MT (32 min), and melatonin (36 min) were identified in
chloroform extracts (Fig. 2A). HPLC analysis of the
aqueous fraction from the same experiment showed the presence of a
major radioactive peak corresponding to serotonin, the presence of a
radioactive peak corresponding to NAS and the absence of radioactive
peaks at the elution time of 5-MT and melatonin (Fig. 2B). The specificity of these findings were
further confirmed by the absence of radioactive peaks of NAS, 5-MT, and
melatonin in control media from primary cell culture of hamster
amelanotic melanoma cells radiolabeled with 5 µCi of
[
H]serotonin (not shown).
Figure 2:
RP-HPLC analysis of
[H]serotonin metabolites produced by hamster skin
and released into culture medium. Skin biopsies were incubated for 5 h
in the medium containing 5 µCi of
[
H]serotonin and 10 µM forskolin (A and B). The media were chloroform extracted and
chloroform (A) and aqueous (B) phases were separated
by RP-HPLC in the presence of nonradioactive standards (see below), and
the radioactivity in collected fractions was measured by liquid
scintillation spectroscopy. SER, NAS, 5-MT, and MEL show radioactive
peaks coeluting with unlabeled standards of serotonin, N-acetylserotonin, 5-methoxytryptamine, and melatonin,
respectively.
The nature of the other radioactive peaks, including peaks eluting between NAS and 5-MT (30-31 min) and the major hydrophobic peak eluting at 40-42 min has not been determined. The GC/MS characteristic of the peak eluting at 26 min between serotonin and NAS is provided below. We have also attempted to characterize the 40-42-min hydrophobic peak using media from the culture performed in the presence of 100 µM unlabeled serotonin. Preliminary IR-mass spectroscopy suggests a nonaromatic compound (data not shown). This peak was also present in control medium (not shown) despite an absence of detectable transformation of serotonin into melatonin. We suggest that this peak may be unrelated to synthesis and degradation of melatonin and, therefore, we have narrowed our HPLC analyses to elution times of the serotonin, NAS, 5-MT, and melatonin standards.
The spectrum of the RP-HPLC separation of skin extracts was similar to that obtained from culture media. The NAS, 5-MT, and melatonin peaks were present in the chloroform fraction, while the majority of the radioactivity corresponding to serotonin and NAS remained in the aqueous fraction (Fig. 3). Comparison of Fig. 2and Fig. 3shows that the serotonin metabolites accumulate predominantly in the culture media.
Figure 3:
RP-HPLC analysis of
[H]serotonin metabolites accumulating in the
hamster skin cultured in vitro. Skin biopsies were incubated
for 5 h in the medium containing 5 µCi of
[
H]serotonin and 10 µM forskolin.
The skin biopsies were homogenized, centrifuged, and supernatant was
chloroform extracted. Chloroform (A) and aqueous (B)
phases were separated in the presence of nonradioactive standards as
described in the legend to Fig. 2. SER, NAS, 5-MT, and MEL show
radioactive peaks coeluting with unlabeled standards of serotonin, N-acetylserotonin, 5-methoxytryptamine, and melatonin,
respectively.
Figure 4:
Production of identified by RP-HPLC
[H]N-acetylserotonin,
[
H]melatonin, and
[
H]5-methoxytryptamine by hamster skin cultured in vitro for 12 h. Skin biopsies were incubated in the medium
containing 10 µCi of [
H]serotonin and 10
µM forskolin. The media were extracted and chloroform (A) and aqueous (B) phases were separated by RP-HPLC
in the presence of nonradioactive N-acetylserotonin,
5-methoxytryptamine, and melatonin (MEL).
For GC/MS identification of NAS in skin culture media, an analysis of ion fragments resulting from EI analysis of purified NAS was initially performed. Fig. 5A shows a total ion count chromatograph of a standard preparation of NAS which was derivatized and chromatographed. The mass spectra insert shows ion fragments with m/z 73, 290 (base peak), 303, 362, and 435. A proposed structure of the ion fragments is shown, along with the mass spectra, in Fig. 5B. The fragmentation pattern indicates a molecular ion with trisilylated derivatization. The major ion fragment with m/z 290 results from ion impact fragmentation of the carbon-carbon bond in the alkyl side chain of the indole ring, leaving a disilylated fragment ion. The ion with m/z 303 is consistent with the loss of silylated N-acetyl group. A minor abundance of a mono-desilated ion fragment with m/z 362 is also observed with the corresponding trimethyl silyl ion fragment at m/z 73. Ions with m/z 434 and 435 are consistent with the trisilylated molecular ion and its proteinated form, respectively.
Figure 5: GC/MS identification of NAS. A, total ion count chromatogram and partial mass spectrum of N-acetylserotonin standard material. B, mass spectrum with proposed ion fragment structures for N-acetylserotonin. C, GC/MS analysis of HPLC fraction eluting at 27-29 min by selective ion monitoring. The upper and middle panels show selective ion chromatograms for the m/z range of 290 to 303 and 290 to 362, respectively. The lower panel shows the relative abundance of ions with m/z 290, 303, and 362 for the compound with a chromatographic retention time of 10 min 53 s. D, GC/MS analysis of N-acetylserotonin by selective ion monitoring. The upper and middle panels show selective ion chromatography for the m/z range of 290 to 303 and 290 to 362, respectively. The lower panels shows the relative abundance on ions with m/z 290, 303, and 362 for the compound with a chromatographic retention time of 10 min 53 s.
Based on the mass spectrum analysis of NAS, ions with m/z 290, 303, and 362 were used in the GC/MS identification of NAS in the aqueous layer of the media from skin culture. Results of the GC/MS analysis of the RP-HPLC fraction eluting at 27-29 min are shown in Fig. 5C. The upper and middle panels show selective ion chromatograms for the m/z range of 290 to 303 and 290 to 362, respectively. Both chromatograms show a single peak eluting at a retention time consistent with NAS. In the lower panel is shown the relative abundance of ions with m/z 290, 303, and 362. Fig. 5D shows a parallel GC/MS analysis of pure NAS. The relative abundance of fragment ions with m/z 290, 303, and 362 shown in the lower panel of Fig. 5, C and D, evidence a close agreement of ion ratios for pure NAS and the compound isolated from skin culture media. Thus, the compound isolated from the skin culture media is identified as NAS based upon HPLC retention, gas chromatographic retention, and mass spectral fragmentation ions.
Furthermore, we analyzed from aqueous phase the RP-HPLC fraction eluting at 25-26 min, which corresponds to the second major radioactive peak eluting between serotonin and NAS (Fig. 2Fig. 3Fig. 4). The analysis was performed by EI and CI modes. The major compound in this fraction has a gas chromatographic retention time of 11 min 48 s. The EI mass spectrum resulted in ion fragments with m/z 73, 354, 410, and 426 (base peak). The ion with m/z 426 also predominated in the CI analysis. Although we do not yet have the structural identity of this compound, we propose that it is a product of serotonin metabolism with a molecular weight of 425 daltons for the derivatized compound. The mass spectrum of the unknown compound was not consistent with the structures of 5-hydroxyindole acetaldehyde, 5-hydroxytryptophol, 5-hydroxyindole acetic acid, 5-methoxytryptophol, and 5-methoxyindoleacetic acid.
Figure 6:
Time-dependent production of
[H]N-acetylserotonin,
[
H]melatonin, and
[
H]5-methoxytryptamine by hamster skin. Skin
biopsies were incubated in the medium containing 10 µCi of
[
H]serotonin. After defined time periods cultures
were terminated, media were extracted, and chloroform phases were
separated by RP-HPLC in the presence of nonradioactive standards of
serotonin (SER), NAS, 5-MT, and melatonin (MEL). The
results are the summary of two skin cultures. A, 3 h of
incubation; B, 5 h of incubation; C, 24 h of
incubation; D, summary panel.
The data from representative RP-HPLC separations performed on the chloroform phase obtained after 6 h of incubation in the absence or presence of 1, 10, and 100 µM forskolin are presented in Fig. 7, A-E. The addition of forskolin stimulated production of tritiated NAS, melatonin, and 5-MT in a dose-dependent manner with a maximal stimulation at 10 µM concentration (Fig. 7C). In a separate experiment we analyzed the aqueous fraction by a RP-HPLC. It appeared that the NAS concentration in the aqueous fraction was higher in the presence of 10 µM forskolin than in control (not shown), which is consistent with the data presented in Fig. 7.
Figure 7:
The effect of forskolin on production of
[H]N-acetylserotonin,
[
H]melatonin, and
[
H]5-methoxytryptamine by hamster skin. Skin
biopsies were incubated for 6 h in the medium containing 10 µCi of
[
H]serotonin and different concentrations of
forskolin: none (A, control), 1 µM (B),
10 µM (C), 100 µM (D),
summary panel (E). Chloroform phases of the culture media from
2 skin cultures were separated by RP-HPLC in the presence of
nonradioactive standards of serotonin (SER), NAS, 5-MT, and
melatonin (MEL).
In previous studies with hamster skin, we have identified and
characterized the NAT-2 isozymic form of arylamine N-acetyltransferase that catalyzed the acetylation of
serotonin to NAS, a direct precursor of melatonin, thus suggesting a
non-rhythmic formation of N-acetylserotonin(22) . We
now have direct experimental evidence showing that mammalian skin can
transform [H]serotonin to radioactive metabolites
eluting at the same time as noradioactive NAS, melatonin, and 5-MT
standards. The GC/MS confirmed that coeluting nonradioactive standards
separated by RP-HPLC were serotonin, NAS, melatonin, and 5-MT.
Furthermore, the GC/MS analysis of the fraction eluting at the same
time as NAS confirms the presence of NAS. Therefore, we conclude that
mammalian skin in vivo can transform serotonin into NAS.
Moreover, on the basis of RP-HPLC separation data we postulate that in
the skin the NAS is further transformed to melatonin and suggest that
it is subsequently deacetylated to 5-MT analogous to the metabolic
pathway in the retina(8, 9, 10) .
According to our estimation, the production of NAS is comparatively
higher than its further transformation to melatonin, i.e. approximately 1-2% of [H]serotonin
added is transformed to [
H]NAS, of which less
than 10% is metabolized further to [
H]melatonin
and [
H]5-MT. This may be explained by a lower
efficiency of the hydroxyindole-O-methyltransferase in
transforming NAS to melatonin and by existence of an alternative
pathway directly metabolizing NAS. The rate at which
hydroxyindole-O-methyltransferase changes in response to
stimuli is relatively slow as compared to that of the arylalkylamine
NAT(24, 25) .
In the pineal gland and retina the
generation of melatonin production exhibits a circadian rhythm with the
peak activity at night(1, 2) . This process is
accompanied by an increased activity of the rate-limiting enzyme in
melatonin synthesis, arylalkylamine NAT that acetylates serotonin to
NAS(1, 2, 3) . These processes are stimulated
by the rise in the intracellular concentrations of
cAMP(1, 2, 3) . It is of interest that in
skin culture the highest production of [H]NAS and
[
H]melatonin was observed at 10 and 12 p.m. and
that this process appeared to be stimulated by forskolin (stimulator of
intracellular cAMP
accumulation(1, 2, 3, 4) ).
Therefore, it is possible that a mechanism governing cutaneous
production of NAS and melatonin may be similar to that operating in the
pineal gland and retina. This requires further study.
In
non-mammalian vertebrates, degradation of melatonin at the site of its
action such as retinal pigment epithelium and skin, or at site of its
extrapineal production, retina, occurred predominatly via melatonin
deacetylation to 5-MT(8, 9, 10) . In the
present study we show the gradual increase of
[H]5-MT during skin culture, which reached its
highest level after 24 h of incubation, and which was accompanied by a
decrease in [
H]NAS and
[
H]melatonin concentration. Furthermore, it was
reported previously that rodent skin is a site for melatonin
bioregulation(20) . Based on this information and the data
presented above we suggest that rodent skin, similar to non-mammalian
skin and retina(8, 9, 10) , deacetylates
melatonin to 5-MT in order to terminate its action or to generate
methoxyindoles with a potential local biological activity.
Previously Finocchiaro et al.(11) showed melatonin biosynthesis and metabolism in peripheral blood leukocytes, while Huether et al.(12) have suggested a possible melatonin synthesis in the gut. This last finding, however, was disputed by others(26) . Presented here are data supporting the concept that there are extracranial and peripherially located sites of melatonin synthesis (11) . Since the integumentum has the same embryonal origin as the central nervous system(27) , it is not surprising that skin is capable of producing neurohormones and expressing corresponding receptors(15, 28) . Skin is composed of many unrelated cells of neuroectodermal and mesenchymal origin including melanocytes, Merkel cells, keratinocytes, resident, and circulating immune response associated cells, fibroblasts, endothelial cells, and fat cells(13, 16, 18, 27) . Future experiments with in situ hybridization techniques using molecular probes from the recently cloned gene coding arylalkylamine NAT (29, 30) may help identify cutaneous cells producing melatonin and may better define the role of melatonin in skin function.
In conclusion, we show that the mammalian skin can produce NAS, melatonin, and 5-MT and suggest that production of 5-MT may represent a local mechanism of melatonin inactivation. Thus, the skin appears to be both a target for melatonin bioregulation and a site of its synthesis and degradation.