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Endocrinology Vol. 147, No. 7 3235-3242
Copyright © 2006 by The Endocrine Society

Characterization of Human Melatonin Synthesis Using Autoptic Pineal Tissue

Katrin Ackermann, Roman Bux, Udo Rüb, Horst-Werner Korf, Gerold Kauert and Jörg H. Stehle

Institute of Anatomy III (K.A., J.H.S.), Institute of Forensic Medicine (R.B., G.K.), Institute of Anatomy I (U.R.), and Institute of Anatomy II (H.-W.K.), Johann Wolfgang Goethe-University, D-60590 Frankfurt, Germany

Address all correspondence and requests for reprints to: Dr. Jörg H. Stehle, Dr. Senckenbergische Anatomie, Institute of Anatomy III, Johann Wolfgang Goethe-University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany. E-mail: stehle{at}em.uni-frankfurt.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mammalian pineal gland synthesizes rhythmically the hormone melatonin, which provides the body with a signal coding the duration of the night period. The ultimate enzymatic step in melatonin synthesis is achieved by the hydroxyindole O-methyltransferase (HIOMT); the rate-limiting enzyme is, however, the arylalkylamine N-acetyltransferase (AA-NAT). In contrast to the central importance of a transcriptional regulation of the Aa-nat gene for rodent melatonin synthesis, mechanisms in the human pineal gland are elusive. Therefore, pineal tissue, taken from regular autopsies (n = 69; postmortem intervals ranging from 9 to 147 h) was analyzed simultaneously for Aa-nat and Hiomt mRNA levels by PCR, AA-NAT activity using 14C-acetyl-coenzyme A, HIOMT activity using S-adenosyl-L-[14C]-methionine, and melatonin content using an ELISA. Results were allocated to asserted time-of-death groups (day, 1000 to 1630 h; dusk, 1630 to 2200 h; night, 2200 to 0730 h; dawn, 0730 to 1000 h). RNA degradation rates of genes of interest ran in parallel, and, therefore, data normalization could be established, regardless of postmortem delay in tissue sampling. Aa-nat and Hiomt mRNA and HIOMT activity showed no diurnal rhythm. In contrast, a significant rhythm was found for the correlation between time of death and both AA-NAT activity and melatonin content, with elevated values during dusk and night. Presented data demonstrate that postmortem brain tissue can be used to detect the remnant of premortem adaptive changes in neuronal activity. In particular, our results give strong experimental support for the idea that transcriptional mechanisms are not dominant for the generation of rhythmic melatonin synthesis in the human pineal gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, AN endogenous oscillator with a genetically encoded circadian (e.g. ~24 h) period is located in the hypothalamic suprachiasmatic nucleus (SCN). The SCN integrates operating experience on the photoperiodic history to mirror and precisely anticipate environmental lighting conditions (1, 2). Of utmost importance is the dissemination of circadian time cues from the SCN to other cells, peripheral tissues, and organs for proper coordination of activity rhythms. One well-described example of such a coordination is the neuroendocrine transduction of rhythmic SCN output by the pineal gland (3). In all vertebrates investigated so far, the pineal gland serves these time-coding needs by a rhythmic synthesis of melatonin, with nocturnally elevated levels dynamically adapting to changes in the duration of the dark period (3, 4).

In mammals, the nocturnally elevated release of the sympathetic neurotransmitter norepinephrine drives through the cAMP-signaling pathway the N-acetylation of serotonin by the arylalkylamine N-acetyltransferase (AA-NAT). Subsequently, N-acetylserotonin is converted by the hydroxyindole O-methyltransferase (HIOMT) to melatonin (4). The gating of the melatonin signal is a conserved similarity between species, determined by the rhythmic activity of the penultimate enzyme in production of this hormone, the AA-NAT. Molecular mechanisms behind the AA-NAT activity rhythm are, however, remarkably different between species (5).

Regulation of the AA-NAT activity is best studied in rodents (5), in which the pivot for the circadian rhythm in melatonin synthesis is a more than 100-fold increase in Aa-nat transcription at nighttime (6, 7). Transcriptional regulation is efficiently flanked by posttranslational mechanisms (8, 9).

In contrast, in all ungulates studied so far (sheep, bovine), Aa-nat mRNA levels do not show significant differences between day and night (10, 11), resulting in a constitutive up-regulation of AA-NAT protein, which is, however, rapidly degraded via proteasomal proteolysis during daytime (4, 5, 10). By transferring results from in vitro experiments (12, 13), it was concluded that the nocturnal increase in AA-NAT activity and melatonin content in the ungulate pineal gland (10, 11) is attributable to a norepinephrine-induced inhibition of proteasomal degradation of this enzyme.

Knowledge on mechanisms of melatonin synthesis in primates, including man, is sparse. In the rhesus monkey Macaca mulatta, no differences were observed between day and night in rhesus Aa-nat and Hiomt mRNA levels, whereas AA-NAT activity and AA-NAT protein showed 10-fold higher nighttime values compared with daytime (14). The therefore suggested posttranscriptional control of the AA-NAT in this monkey is supported by the fact that melatonin values in M. mulatta cerebrospinal fluid increase without a lag period shortly after lights off (15) and rapidly decrease on light exposure at nighttime (16).

Analysis of molecular mechanisms behind the melatonin synthesis in man are for obvious reasons more difficult. The rhythmic profile of human melatonin synthesis can easily be assessed using salivary, plasma, or urine samples and is truly of circadian nature because it persists even in the absence of a light-dark cycle (17). Rhythm characteristics are highly reproducible from day to day in the same individual, but with large interindividual differences in absolute values (17). In humans, the melatonin profile in body fluids shows a rapid dim-light melatonin onset (18) at the beginning of the night, and a suppression of nocturnally elevated hormone synthesis occurs already during application of low-light intensities (19, 20).

Insight into the molecular mechanism driving rhythmic melatonin synthesis in the human pineal gland can currently only be achieved by using postmortem tissue. The feasibility of such an approach was demonstrated by finding a remnant profile of a melatonin rhythm in postmortem material (21, 22) that correlates significantly to the diurnal profile obtained in healthy men (17, 23). In addition, using autoptic human pineal tissue, residual AA-NAT and HIOMT enzyme activities were demonstrated (23), and no significant differences were found between day and night for Aa-nat and Hiomt mRNA levels (24).

The melatonin profile in humans and the activity and/or structure of the elements of the synthesis pathway of the hormone are often altered in some pathophysiological states (25, 26). To use melatonin in preventive or curative interference with the human circadian system, a complete understanding of the generation of the rhythmic hormonal signal in the pineal gland is warranted. We therefore analyzed in pineal glands, taken from regular autopsies Aa-nat and Hiomt mRNA content, AA-NAT and HIOMT enzyme activities and melatonin levels at the same time and within the same subject.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue sampling
All experiments are in accordance with The Declaration of Helsinki, and experiments were formally approved by the local ethics commission of the University Clinics Frankfurt/Main, Germany. All autopsies were conducted as a consequence of an obligatory assignment by federal prosecutor and German law. Before autopsy, the human bodies (n = 69; for details, see supplemental data published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) were kept at 4–8 C after transfer to the Institute of Forensic Medicine. During mandatory autopsies, the scull was opened, and adhesive meninges (dura mater, arachnoid membrane) were removed. The brain was cut horizontally (Flechsig cut), the pineal stalk was dissected, and the pineal gland was removed, fractionalized into several pieces, and stored individually at –80 C until further use.

Time of death was appointed, according to the routine postmortem inspection, as documented in the death certificate. Postmortem interval (PMI) (time between death and pineal excision) ranged between 9 and 147 h (mean, 47 ± 4 h).

Of the 69 pineal glands collected, five were excluded from analysis because of severe damage to brain tissue (n = 3), as detected during autopsy, or because of a determination of time of death that exceeded our analysis limits (n = 2), and three pineal glands were used exclusively for a degradation experiment (see below).

The age of the analyzed subjects ranged between 10 and 84 yr (mean, 46.4 ± 2.1 yr; age distribution, <20 yr, n = 1; 21–30 yr, n = 8; 31–40 yr, n = 17; 41–50 yr, n = 9; 51–60 yr, n = 14; 61–70 yr, n = 5; 71–80 yr, n = 5; 81–90 yr, n = 2) and did not vary significantly between males and females (male, n = 40, mean of 45.0 ± 2.4 yr; female, n = 21, mean of 49.1 ± 4.2 yr). Causes of death were intoxication (n = 17), heart diseases (n = 17), bleeding to death (n = 6), traffic accidents (n = 3), strangulation (n = 2), pneumonia (n = 2), purler (n = 2), hanging (n = 2), multiorgan failure (n = 2), drowning (n = 2), brain death (n = 1), decapitation (n = 1), aspiration (n = 1), hypoxia (n = 1), circulatory collapse (n = 1), and undetermined (n = 1) (for more details, see supplemental data published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

Pineal glands were allocated to four groups, according to the appointed time of death, with a day group (1000 to 1630 h; n = 11), a dusk group (1630 to 2200 h; n = 18), a night group (2200 to 0730 h; n = 22), and a dawn group (0730 to 1000 h; n = 10). Bin widths of the four time groups were chosen on the basis of epidemiologic coincidence with melatonin onset and offset (17) and a season-independent gating of the daytime interval.

As an internal control, an analysis of dynamics in protein and mRNA degradation was performed using three additional complete human pineal glands selected from different time groups. These samples were kept on autopsy at 4 C to study degradation processes under controlled experimental conditions.

RNA isolation, RT, and PCR
RNA isolation and RT.
Total RNA from human pineal fractions was isolated (Absolutely RNA miniprep kit; Stratagene, La Jolla, CA), as described previously (27). A random-primed RT was performed for 1 h at 37 C, with 1 µg total RNA, 1.5 µl 10x hexanucleotide mixture (Roche, Penzberg, Germany), 200 U Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI), 5 µl Moloney murine leukemia virus RT reaction buffer, and 1 µl dNTPs (10 mM, 2.5 mM each) in a final volume of 25 µl. Obtained cDNA was stored at –20 C.

RT-PCR.
PCR amplification was performed with a Taq DNA polymerase kit (Invitrogen, Carlsbad, CA). Briefly, 1 µl of the RT reaction was mixed with 2.5 µl 10x PCR buffer, 0.75 µl MgCl2 (50 mM), 0.5 µl dNTPs (10 mM, 2.5 mM each), 1 µl of each primer (10 mM), and 0.25 µl Taq polymerase in a final volume of 25 µl.

PCR was performed for Aa-nat, Hiomt, and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) using the following primer sequences: Aa-nat forward, 5'-CAGAGCACCCACCCCCTGAAAC-3'; Aa-nat reverse, 5'-CCTGCATGAGTCTCTCCTTGTC-3', amplifying a 310 bp fragment (GenBank accession no. NM_001088); Hiomt forward, 5'-CGCCTCCTTAATGACTACGCCA-3'; Hiomt reverse, 5'-TTGACGCTCCAGACCTCCTG-3', amplifying a 473 bp fragment (28) (GenBank accession no. NM_004043); Gapdh forward, 5'-GCACCGTCAAGGCTGAGAAC-3'; and Gapdh reverse, 5'-GCCTTCTCCATGGTGGTGAA-3', amplifying a 150 bp fragment (GenBank accession no. NM_002046).

Primers for human Aa-nat and Gapdh were designed using the ABI Prism primer design software (Applied Biosystems, Foster City, CA). Amplification conditions were as follows: first amplification round at 94 C for 5 min, annealing temperature (Ta) for 1 min, and 72 C for 2 min. Subsequent amplification rounds were as follows: 94 C for 45 s, Ta for 1 min, and 72 C for 2 min. Number of cycles and Ta was optimized (data not shown) for each primer pair (Aa-nat, 32 cycles, Ta of 61 C; Hiomt, 35 cycles, Ta of 65 C; Gapdh, 26 cycles, Ta of 61 C).

For each primer pair, a PCR amplification was performed with at least two different cDNA samples. Optimal Ta and cycle numbers were determined by visualizing PCR products on an agarose gel, taking intensity and specificity of the bands as criteria.

PCR products were quantified using an image analysis system (ChemiDoc XRS, Quantity One; Bio-Rad, Hercules, CA) with a mass standard (Bio-Rad). Intensities of Aa-nat, Hiomt, and Gapdh bands, acquired from each pineal cDNA, were digitized, and the calibration curve was applied and, for comparative reasons, expressed as the percentage of the maximal value for a given gel. Values for Aa-nat and Hiomt bands were normalized against corresponding values for bands of the housekeeping gene Gapdh.

Quantitative real-time PCR.
To verify data from RT-PCR analyses, a real-time PCR was performed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with selected samples (n = 11), using the SYBR Green PCR kit (Applied Biosystems), according to the instructions of the manufacturer. PCR was performed with 1 µl cDNA in a final volume of 25 µl in a 96-well plate, with the same Gapdh primers as used in the RT-PCR analyses (see above) and with primer pairs for Aa-nat and Hiomt as described previously (24). Samples were measured in triplicate and obtained values averaged. Data analysis was performed using the 2(-{Delta}{Delta}C(T)) method, which was validated according to the manufacturer (see the ABI Prism User Bulletin 2; Applied Biosystems).

Preparing pineal homogenates
For determination of AA-NAT activity and melatonin content, tissue homogenates of pineal fractions were sonicated in 0.1 M ammonium acetate buffer (pH 6.8) containing protease (Protease Inhibitor Cocktail Tablets; Roche) and phosphatase (Phosphatase Inhibitor Cocktail Set II; Calbiochem, San Diego, CA) inhibitors and centrifuged at 14,000 rpm for 15 min at 4 C. Homogenates for HIOMT activity assay were prepared using 0.1 M phosphate buffer (pH 7.9). Supernatants were removed, and protein concentrations were determined using a Protein Assay kit (Pierce, Rockford, IL), with BSA as a standard, according to the instructions of the manufacturer. Supernatants were stored at –80 C.

AA-NAT and HIOMT activity assay
With prepared homogenates from human pineal tissue, none of the commercially available AA-NAT antibodies gave a reproducible band of expected size in immunoblot experiments in our hands (data not shown). However, using the same homogenates, AA-NAT activity could be reliably determined using a protocol as described previously (29). Briefly, homogenates (20 µl per sample) were incubated at 37 C in an ammonium acetate buffer (pH 6.8) containing tryptamine (1 mM; Fluka, Buchs, Switzerland) and 14C-acetyl coenzyme A (10 mM; specific activity, 60 Ci/mol; PerkinElmer, Boston, MA).

For HIOMT activity, homogenates (20 µl per sample) were incubated at 37 C in phosphate buffer (pH 7.9), containing N-acetyl-serotonin (1.3 mM; Sigma-Aldrich, Steinheim, Germany) and S-adenosyl-L-[14C]-methionine (0.06 mM; specific activity, 55 Ci/mol; PerkinElmer). The organic phase was washed twice with sodium borate buffer (20 mM; pH 10), chloroform extracted, and dried.

For both AA-NAT and HIOMT activity, incorporated radioactivity was measured for each subject in duplicate, averaged, and normalized to protein content.

Melatonin assay
Melatonin content in pineal tissue was determined in duplicate for each sample and with two dilutions (1:50 and 1:500) with an ELISA kit (IBL, Hamburg, Germany). Homogenized samples were diluted in ammonium acetate buffer (pH 6.8), and melatonin was extracted according to the protocol of the manufacturer. Signals were densitometrically analyzed with an ELISA reader (Multiscan RC; LabSystems, Altringham, UK). Melatonin content was calculated against standard values provided by the manufacturer and normalized to protein content.

Statistical analysis
The Kruskal-Wallis H test was applied to determine whether the time of death of the subjects (allocated to the four different groups: day, dusk, night, and dawn) was associated to AA-NAT or HIOMT activity or to melatonin content, respectively. Significant associations were further characterized by means of a nonparametric trend analysis. Gene expression data (RT-PCR, real-time PCR) of the four groups were analyzed using a one-way ANOVA and a subsequent Bonferroni’s test, with P < 0.05 as the criterion of significance.

Differences in proportion between male and female subjects within the four groups were tested using the {chi}2 test. The Kolmogorov-Smirnov test for normality was applied to test the distribution of age and PMI within the four groups.

To exclude the influence of confounding factors (age, gender, PMI) on the parameters analyzed, the Mann-Whitney U test (gender) was performed, and correlations were analyzed using the Spearman’s rank correlation coefficient {rho} (age, PMI), with P < 0.05 as the criterion of significance (one-tailed).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
All obtained data were allocated to the four time-of-death groups (night, dawn, day, and dusk). Application of the {chi}2 test showed no differences in proportion between male and female subjects within the four groups ({chi}2 = 2.57; df = 3; P > 0.49), and application of the Kolmogorov-Smirnov test revealed a normal distribution of age and PMI in all four groups (all P values > 0.2).

In addition, calculation of the Spearman’s rank correlation coefficient {rho} revealed no significant correlations between potentially confounding factors (age, PMI) and the parameters of interest in the four time groups (all P values > 0.1). Finally, according to the results of the Mann-Whitney U test, experimental results were independent of the gender of the individuals under consideration (all P values > 0.25).

RNA analyses
Aa-nat and Hiomt mRNA degradation experiment.
The amount of extracted total RNA, maintained at 4 C constant conditions, did not vary significantly with incubation time, as assessed by spectrophotometry (data not shown). However, when applying a highly sensitive PCR amplification on extracted RNA, a clear decline with time in mRNA levels became evident (Fig. 1AGo), with slopes of regression lines for Gapdh (y = –1.3874x + 95.302), Aa-nat (y = –1.5648x + 90.328), and Hiomt (y = –1.0604x + 70.04) being remarkably similar (Fig. 1BGo). Extending regression lines indicate a total absence of intact RNA, at the latest, after 70 h under these artificial conditions. This decline in RNA content is largely accelerated compared with the situation in the corpse, in which intact RNA could still be recovered at a PMI of 141 h.


Figure 1
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  		FIG. 1. RNA degradation with time in human pineal tissue. A, RT-PCR analyses of signal intensities achieved with specific primers for Aa-nat, Gapdh, and Hiomt. Depicted are amplification products obtained when tissue samples were maintained for indicated time intervals at 4 C. Note the loss of signal intensities with time. To the right of each of the representative gel pictures, the expected band size is indicated. B, Semiquantitative densitometric analyses of signals for individual samples, as shown in A, with a linear regression curve fitted to obtained values. Note the similar decline in the signal intensities, representing amounts of amplification products with time.

 
Aa-nat and Hiomt mRNA levels in autoptic pineal tissue.
Because of tissue availability, intact mRNA could be extracted from 55 of the 61 pineal glands processed. Large interindividual differences in absolute values for amounts in amplification products for a given gene were detected (data not shown). However, mean RNA amounts for both Aa-nat and Hiomt did not show significant differences between the four time-of-death groups when normalized against Gapdh (Fig. 2Go). This observation was confirmed in a real-time PCR analysis with randomly selected samples from each time-of-death group (data not shown).


Figure 2
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  		FIG. 2. Diurnal analyses of relative Aa-nat and Hiomt mRNA levels in human pineal tissue. Standardized RT-PCR analyses were applied to RNA, extracted from human pineal tissue (n = 55). Obtained signals for Aa-nat and Hiomt amplification products were normalized against corresponding Gapdh values. Results were allocated to the four groups (night, n = 21; dawn, n = 8; day, n = 11; dusk, n = 15) according to time of death of each individual subject. Depicted are the means ± SEM of the relative mRNA values. No significant correlation was found between mean values for Aa-nat and Hiomt mRNA and time of death.

 
Analyses of AA-NAT and HIOMT activity
PMI had only a marginal influence on the decay of specific protein signals in pineal tissue (P > 0.31 for actin, and data not shown), confirming previous observations with autoptic human brain material (30, 31).

During the analyses, it turned out that HIOMT activity is by far the most reliable parameter to characterize the remnant biochemical integrity of pineal tissue. Analysis of subjects showed that the decline in HIOMT activity with increasing PMI is much slower compared with the decay of AA-NAT activity (data not shown). Based on this observation, together with the previously reported superior stability of HIOMT activity [halving time, ~24 h (32)] compared with AA-NAT activity [halving time, ~3 min (33)], we excluded those subjects from statistical analysis, which showed no or very low levels of HIOMT activity (<100 fmol/h/µg protein; n = 10). Indeed, subsequent analyses showed no or very low levels of AA-NAT activity and melatonin content exactly in these samples, regardless of time of death of subjects.

AA-NAT activity.
AA-NAT activity was measured in 61 human pineals, of which 10 samples with no or very low activity were excluded from additional analysis because they also showed very low or absent levels of HIOMT activity (see above).

Although the number of samples investigated in the dusk (n = 13) and in the night group (n = 20) outnumber by far the sample number in the dawn (n = 8) and day (n = 10) groups (Fig. 3Go), pineals with undetectable AA-NAT activity were notably higher in number in the latter groups (dawn/day, n = 12 vs. dusk/night, n = 7). High values of AA-NAT activity (>51 fmol/h/µg protein content; n = 6) were only present in the dusk and night groups (Fig. 3AGo).


Figure 3
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  		FIG. 3. Diurnal analysis of AA-NAT activity in autoptic pineal tissue. A, Distribution of AA-NAT activity values within the four groups (night, dawn, day, and dusk) allocated to five ranges in enzyme activity. Note the exclusive appearance of high and highest AA-NAT activity (>26 fmol/h/µg protein) in the dusk and night groups. Number of samples that were allocated to the different range groups according to the measured AA-NAT activity is indicated at the right of each column. B, Double plot of the diurnal pattern of AA-NAT activity, depicted as mean ± SEM AA-NAT enzyme values for each of the four time-of-death groups. A significant quadratic correlation was found between mean values for AA-NAT activity and time of death.

 
AA-NAT activity values were significantly correlated to time of death (Hcorr = 9.0330; P < 0.05). Subsequent nonparametric trend analysis revealed a significant quadratic trend for the correlation between AA-NAT activity and time of death (Hquad = 7.4965; P < 0.01) (Fig. 3BGo).

HIOMT activity.
Because of a restriction in tissue availability, HIOMT activity could not be analyzed in 18 of 61 pineal glands. However, in these 18 samples, all other parameters were determined.

Measured HIOMT values were uniformly distributed with respect to time of death (Fig. 4Go).


Figure 4
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  		FIG. 4. Diurnal analysis of HIOMT activity in autoptic pineal tissue. A, Distribution of HIOMT activity values within the four groups (night, dawn, day, and dusk) allocated to four ranges in enzyme activity. Number of samples that were allocated to the different range groups according to the measured HIOMT activity is indicated at the right of each column. B, Double plot of the diurnal pattern of HIOMT activity depicted as mean ± SEM HIOMT enzyme values for each of the four time-of-death groups. No significant correlation was found between mean values for HIOMT activity and time of death.

 
Determination of melatonin content
Melatonin was measured in the same 61 human pineal samples as analyzed for AA-NAT activity. In those 10 samples with very low or undetectable HIOMT activity (see above), no or very low amounts of melatonin could be detected, and samples were excluded from additional analysis. Highest melatonin levels (>1000 pg/mg protein) were found only in the nighttime group. Notably, of the 30 subjects with undetectable or very low values (0–100 pg/mg protein), the smallest group (n = 6) was present in the group with the highest number of samples, the night group (n = 20) (Fig. 5AGo).


Figure 5
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  		FIG. 5. Diurnal analyses of melatonin content in autoptic pineal tissue. A, Distribution of melatonin values within the four groups (night, dawn, day, and dusk) allocated to four ranges in hormone values. Note that, although the night group is the largest of all time-of-death groups (n = 20), the sample number with low to undetectable melatonin levels is the smallest (n = 6) in this group and that highest melatonin values appear exclusively in this time-of-death group. Number of samples that were allocated to the different range groups according to the measured melatonin content is indicated at the right of each column. B, Double plot of the diurnal pattern of measured melatonin levels depicted as mean ± SEM hormone values for each of the four time-of-death groups. A significant quadratic correlation was found between mean values for melatonin content and time of death.

 
Time of death was highly significantly correlated with melatonin content (Hcorr = 10.7135; P < 0.025) (Fig. 5BGo). Additional nonparametric trend analysis revealed a significant quadratic trend for this correlation (Hquad = 4.0650; P < 0.05) (Fig. 5BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Melatonin synthesis is one of the best available measures to analyze the functional state of the endogenous circadian clock in the mammalian SCN. In humans, the molecular mechanisms underlying the generation of the melatonin message of darkness can currently only be deciphered using autoptic material. Here presented data show the feasibility of this approach and analyze Aa-nat and Hiomt mRNA levels, AA-NAT and HIOMT activity, and melatonin content from human postmortem pineal tissue simultaneously in the same subjects. Results depict for the first time a clear diurnal rhythm in AA-NAT activity and melatonin content, despite constant values for Aa-nat and Hiomt mRNA and for HIOMT activity. Thus, results demonstrate that, for the generation of the circadian melatonin profile in the human pineal gland, posttranscriptional/translational mechanisms seem to be central.

The analysis of mRNA decay initially performed here shows that autoptic pineal tissue can be treated as an ordered system, with an almost parallel decline in levels of different mRNAs over time. In addition, our data indicate the persistence of possible endogenous, daily variations in a given mRNA after death. Furthermore, these observations confirm the general assumption that RNAs are much more vulnerable in human autoptic brain tissue compared with the influence of PMI on protein degradation, which seems to be of only minor importance (30, 31) (Ackermann, K., and J. H. Stehle, unpublished observations). The validity of using autoptic brain material, with analysis conducted with variations in PMI and unknown premortem lifestyle, has been demonstrated previously (34, 35, 36). Indeed, whereas obtained mRNA values for Aa-nat (and Hiomt) did not fluctuate over a 24 h day-night cycle, AA-NAT activity shows a highly rhythmic pattern, with elevated levels preceding the observed increase in pineal melatonin content.

In our hands, RNA analysis in autoptic brain tissue by RT with random hexamers is the method of choice (Ackermann, K., and J. H. Stehle, unpublished results) (37) because this method remains rather unaffected by possible changes of RNA structure (31, 34). Based on the molecular integrity of mRNA in autoptic pineal tissue, it can be concluded that the absence of diurnal variations in elevated Aa-nat and Hiomt mRNA levels over time is indeed a remnant silhouette of the in vivo situation. These findings give strong evidence for a regulation of human melatonin synthesis, which is independent of a rhythmic transcription, a fact that has been discussed previously (24). Similarly, variations in Aa-nat mRNA levels between day and night were reported to be negligible in M. mulatta (14). Evidence for a diurnal posttranscriptional regulation of the human AA-NAT were also suggested on the basis of in vitro biochemical analyses of this enzyme (38) using the human Aa-nat clone (10): elevated AA-NAT protein levels were shown to be independent of Aa-nat transcription and resulted from a shielding of the constitutively synthesized enzyme from active dephosphorylation and subsequent proteolysis by forming a stable AA-NAT/14-3-3 protein complex (13, 39).

Here presented data show that, despite an undetectable rhythm in Aa-nat and Hiomt mRNA values over the four death time groups, AA-NAT activity is profoundly elevated in the dusk and night periods compared with dawn and daytime. Thus, we demonstrate for the first time a rhythm in AA-NAT activity in the human pineal gland. Elevated values for AA-NAT activity during dusk and nighttime in the autoptic pineal material are approximately 1000 times lower than those reported for the primate M. mulatta (14). This finding can be explained by ongoing degradation/loss of enzymatic integrity of the AA-NAT protein, likely caused by a halving time in rat of approximately 3 min (33). However, the validity of the AA-NAT rhythm is strengthened by the fact that the ultimate enzyme in melatonin synthesis, the HIOMT, does not show any fluctuation over time, despite its higher stability compared with AA-NAT (see Results) (32).

The elevated AA-NAT activity during dusk presented here does not correspond with a simultaneous elevation in melatonin content. It is currently unclear whether this finding may suggest an important role of the ultimate enzyme in melatonin synthesis, HIOMT, for rate-limiting rhythmic hormone production, as demonstrated recently in the rodent pineal gland (40, 41). It also may be envisioned that this time gap between elevated AA-NAT activity and hormone synthesis is required for an intracellular translocation of N-acetylserotonin, the product of AA-NAT enzymatic activity, from its synthesis place to the place of methylation by the HIOMT. This assumption is based on the fact that large differences exist in the optimal pH for AA-NAT (pHopt 6.8) and HIOMT (pHopt 7.9) activity (42 , and references therein), making it economically unreasonable for pinealocytes to let the two enzymes work together in the same cellular compartment at suboptimal pH conditions. However, the general diurnal silhouette of AA-NAT activity, as assessed here from postmortem pineal glands, shows almost perfect parallel dynamics to observed melatonin levels.

Nighttime melatonin levels were highly significantly correlated to time of death, despite the long time stretch of the night interval chosen here (2200 to 0730 h), with borders corresponding to the onset and offset of hormone secretion in temperate zones (17). Nighttime melatonin values in the postmortem material showed large interindividual differences, matching several previous case studies (17, 20, 23). In addition, our findings explain the rapid dim-light melatonin onset in humans because no change in gene expression is required for up-regulation of melatonin synthesis.

Together, the data presented here (Fig. 6Go). strongly support the view that a posttranscriptional regulation of the AA-NAT protein is the dominant mechanism to generate rhythmic melatonin synthesis in the human pineal gland and thus may be common to all primates.


Figure 6
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  		FIG. 6. Melatonin synthesis in the human pineal gland: a schematic pattern. Schematic presentation of Aa-nat and Hiomt mRNA, AA-NAT and HIOMT activity, and melatonin content adapted to data points of the four time-of-death groups as obtained from autoptic human pineal tissue. For clarity, relative values are double plotted against asserted time of death.

 


   Acknowledgments
 
Members of the Institute for Forensic Medicine, University Frankfurt, and, in particular, the assistants performing autopsies, namely H. Auer, S. Eckhold, and M. Enders, are thanked for their continuous help and support during tissue collection. We thank M. Karolczak, C. Schomerus, G. Burbach, E. Ghebremedhin, E. Maronde, G. Sties, B. v. Schemm, S. Lesny, J. Babica, M. Bernard for primer gift (Hiomt), and M. Sachs for help.


   Footnotes
 
This work was supported by Arthur-und-Margarete-Ebert-Stiftung.

None of the authors has any potential conflicts of interest with data published in this manuscript.

First Published Online March 23, 2006

Abbreviations: AA-NAT, Arylalkylamine N-acetyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIOMT, hydroxyindole O-methyltransferase; PMI, postmortem interval; SCN, suprachiasmatic nucleus; Ta, annealing temperature.

Received January 12, 2006.

Accepted for publication March 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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