Daily rhythms of norepinephrine, β 1 -adrenoceptor mRNA, serotonin, arylalkylamine N...

Daily rhythms of norepinephrine, b1-adrenoceptormRNA, serotonin, arylalkylamine N-acetyltransferasemRNA, arylalkylamine N-acetyltransferase andhydroxyindol-O-methyltransferase activities, andmelatonin in the pineal gland of viscacha

CLAUDIA CALDERON1, LUCIA FUENTES1, ESTELA MUNOZ2,

MORTEN MØLLER3, & LILIAN PELZER1

1Farmacologıa, Universidad Nacional de San Luis, 5700 San Luis, Argentina, 2National Council of

Research, Science and Technology (CONICET) and Instituto de Histologıa y Embriologıa (IHEM),

Mendoza, Argentina and 3Institute of Medical Anatomy, University of Copenhagen, Denmark

AbstractWe described the daily rhythms of parameters involved in melatonin production in the pineal ofviscacha. Norepinephrine showed the highest levels at night. A peak in b1-adrenoceptor mRNA wasobserved at the end of the light phase. Serotonin exhibited higher values during the light period.AA-NAT mRNA showed the highest expression at Zeitgeber time 0 (ZT0), reaching a minimum atnight. AA-NAT activity and melatonin content exhibited a nocturnal peak at ZT16, a lower diurnalincrease during the light period and minimal level at ZT8. HIOMT activity showed a peak at ZT16. Thedaily pattern in norepinephrine content was similar to that of AA-NAT activity and melatonin levels.Our data suggest that the melatonin synthesis in viscacha might be activated via adrenergic induction;the differences observed in the daily rhythms of AA-NAT activity and its mRNA suggest that post-transcriptional mechanisms may be involved, and other Zeitgeber and other mechanisms might alsocontrol melatonin production.

Keyswords: Daily rhythm, pineal gland, melatonin, viscacha, Lagostomus maximus maximus

Introduction

The pineal gland is a neuroendocrine transducer of daily and seasonal time, via the nocturnal

synthesis and secretion of melatonin (Goldman 2001).

The photic information is conveyed from the retina to the pineal through a complex

nervous pathway which includes the circadian pacemarker located in the suprachiasmatic

nuclei of the hypothalamus (SCN) and the superior cervical ganglia that innervate the pineal

gland (Moore 1996). The circadian synthesis of the hormone melatonin is established by

postganglionic sympathetic fibres and their neurotransmitter norepinephrine (NE). However,

Correspondence: Claudia Patricia Calderon, Farmacologıa, Facultad de Quımica, Bioquımica y Farmacia, Universidad Nacional de

San Luis, Chacabuco y Pedernera, 5700 San Luis – Argentina. Tel: 54 2652 424689 (internal 162). Fax: 54 2652 431301.

E-mail: [email protected]; [email protected];[email protected]

Biological Rhythm Research

April 2008; 39(2): 93 – 107

ISSN 0929-1016 print/ISSN 1744-4179 online � 2008 Taylor & Francis

DOI: 10.1080/09291010701324715

other neurotransmitters and certain peptides present in the pineal gland may also be involved

in this process (Møller 1999; Møller et al. 1996, 1997a, 1998, 1999; Korf et al. 1998; Pevet

et al. 1994; Simonneaux et al. 1996, 1997, 1998; Ribelayga et al. 1998; Simoneaux &

Ribelayga 2003). Nocturnal NE release leads to an induction of cAMP formation via b1- and

a1-adrenoceptors resulting in an enhanced transcription of the AA-NAT gene (Klein et al.

1992; Drijfhout et al. 1996).

Møller et al. (1997b) have demonstrated that b1-adrenoceptor mRNA is present in the

pineal parenchyma and exhibits diurnal variations. The increase in b1-adrenoceptor mRNA at

the mid-dark phase involves stimulation of the b1-receptor and is mediated via both

transcriptional and non-transcriptional mechanisms (Carter 1993).

In the pineal gland, melatonin is synthesized from serotonin by arylalkylamine

N-acetyltransferase (AA-NAT) and hydroxyindol-O-methyltransferase (HIOMT) enzymes.

Melatonin rhythm is mainly dictated by the penultimate enzyme in the melatonin biosynthetic

pathway, AA-NAT (Klein & Weller 1970; Iuvone & Besharse 1983; Zawilska & Iuvone

1989). A large increase in the AA-NAT activity during the darktime may reflect both

transcriptional and post-transcriptional events triggered by the neurotransmitter NE. The

importance of transcriptional control of the AA-NAT activity varies among vertebrates. This

mechanism is dominant in rodents like the rat; post-translational modifications of the

AA-NAT protein is the central regulatory mechanism in ungulates like sheep, bovine, and

possibly primates (Stehle et al. 2001; Schomerus & Korf 2005). On the other hand, Ribelayga

et al. (2000) suggested an important physiological role for HIOMT in the photoperiodic

changes related with the amplitude of the nocturnal peak of melatonin in the Siberian

hamster. Ceinos et al. (2004) investigated the adrenergic mechanisms of melatonin synthesis

in the Siberian hamster, suggesting the potentiation of AA-NAT activity by adrenergic

receptor agonists and emphasizing that the rate of melatonin production could be limited

downstream in the metabolic pathway, most probably at the level of HIOMT.

The viscacha (Lagostomus maximus maximus) is a photoperiodic hystricomorphous rodent

with nocturnal habits, belonging to the family Chinchillidae, living in the southern

hemisphere in the mountains or on the pampas. They live in large colonies in extensive

burrows, and the animals emerge at dawn and dusk to feed (Llanos & Crespo 1952). Previous

studies demonstrated that the pineal gland and its hormone, melatonin, are involved in its

annual reproductive cycle characterized by a gonadal regression period during the short

winter days in South America (Fuentes et al. 1991, 1993, 2003, 2004; Domınguez et al. 1987;

Pelzer et al. 1999; Munoz et al. 1997, 1998, 1999, 2001). A daily melatonin rhythm

characterized by a nocturnal peak and a second increase during the light phase was reported in

the retina, pineal gland and serum of viscacha (Calderon et al. 2001). Thus, this wild rodent

might be an interesting model with which to analyze daily rhythms and their control

mechanisms, which have not previously been reported for hystricomorphous rodents.

The objective of the present investigation was to characterize daily rhythms of norepine-

phrine, b1-adrenoceptor mRNA, serotonin, N-acetyltransferase mRNA, N-acetyltransferase

and hydroxyindol-O-methyltransferase activities, and melatonin in the pineal gland of viscacha

(Lagostomus maximus maximus) during the period of maximal gonadal activity.

Materials and methods

Animals and general protocol

Adult male viscachas (Lagostomus maximus maximus), weighing 5 – 7 kg, were used in this

study. All experiments were in compliance with the Administracion Nacional de

94 C. Calderon et al.

Medicamentos y Alimentos y Tecnologıa Medica (ANMAT) N8 6344/96 for Animal

Care Guidelines. The animals were captured in their natural habitat near San Luis,

Argentine (338 200 south latitude, 760 m altitude) during the period of maximal gonadal

activity. The natural light:dark cycle was 11:13 with the normal onset of light period at

ZT0 and offset at ZT11. Four animals were anesthetized with Penthotal (i.p.; 25 mg/kg) at

ZT0, 4, 8, 12, 16 and 20, and a blood sample was obtained from each animal by

cardiac puncture. The serum was separated by centrifugation, transferred to glass vials

and stored at 7208C until assayed. After the animals were sacrificed by decapitation, the

pineal glands were quickly removed and frozen on crushed dry ice and stored at 7808Cuntil use. Pineal glands were cut into 15-mm thick cryostat sections by in situ hibridization

study.

Chemicals

cDNA antisense oligonucleotide probes were obtained by custom synthesis (DNA

Technology, Aarhus, Denmark). The b1-receptor probe was complementary to the base

sequence encoding bases 19 – 63 (CAG CGG CGC GGC CGA CAG GTT GCA GGG

TTC GGA GGC GCC CAG GGC) of the b1-receptor gene of the rat (Machida et al. 1990).

Three cDNA AA-NAT probes were constructed from the arylalkylamine N-acetyltransfer-

ase mRNA clone sequence of Rattus norvegicus (GenBank accession no.U38306; bases:

121 – 159, GGC AGA TGC AGG GCC TCA GGT TTC AGG GGG TGG ATG CTC;

661 – 699, CAC CGT AAG GAA CAT TGC AGC TCA GTG AAG GTG AGA GAT;

1141 – 1179, GTA CCC CAA TGA TGC CAC CAT TCA CAA GTC CCC GGC TCC).

Terminal transferase was obtained from Boehringer Mannheim Biochemica, Germany (no.

220582), 35S-labeled deoxyadenosine 50-(b-thio)triphosphate from DuPont, USA (no. NEG

034H, 37 – 55.5 TBq/mmol).

Acetyl-1-14C coenzyme A (59.2 mCi/mmol) was purchased from New England Nuclear

Corporation, USA. Tritiated Melatonin was obtained from NENTM Life Science Products,

Inc., USA.

Spectrofluorometrical analysis of norepinephrine

The spectrofluorometrical method of Laverty and Taylor (1968) was used. Bryefly, pineal

samples were homogenized in 0.4 N percloric acid (10 ml/g tissue). After centrifugation for

15 min at 5000 rev/min, the supernatant was pippetted. The following reagents were added to

form a fluorescent product: phosphate-EDTA buffer, iodine solution, alkaline sulfite

solutions and glacial acetic acid. The fluorescence was measured in an Aminco-Bowman

spectrophotofluorometer using 1 cm quartz cuvettes. Measurement of excitation (385 nm)

and emission (485 nm) wavelengths was done. Norepinephrine bitartrate monohydrate was

used as the standard. Results were expressed as mg/g tissue.

Oligonucletide 30-labeling

Aliquots of 1 ml oligonucleotide probe (5 pmol/ml) in a solution containing 1 ml terminal

transferase (25 units), 5 ml 35S-ATP, 10 ml reaction buffer (1 M potassium cacodylate,

125 mM TRIS-HCl, bovine serum albumin, 1.25 mg/ml, pH 6.6), and 28 ml sterile water

were incubated for 60 min at 378C. The reaction was stopped by 10 mM TRIS-EDTA

buffer (pH 7.6), then the labeled oligonucleotide was extracted with phenol/chloroform/

isoamyl alcohol (50:49:1) followed by chloroform/isoamyl alcohol (49:1), precipitated in a

Daily rhythms in melatonin-synthesizing pathway 95

NaCl/ethanol solution on dry ice, and dissolved in 100 ml TRIS/EDTA with 0.15% DDT

(dithiothreitol). The specific activity obtained was about 161018 dpm/mol.

In situ hybridization with the 35S-labeled probe

All instruments for in situ hybridization were sterilized before use. RNAase-free diethyl

pyrocarbonate (DEPC)-treated water was used to make up all solutions.

Prior to hybridization, frozen tissue sections were thawed, fixed for 5 min in 4% para-

formaldehyde in phosphate-buffered saline (PBS), and washed 261 min in PBS. The

sections were then acetylated in 0.25% acetic anhydride (diluted in 0.1 M triethanolamine

and 0.9% NaCl ) for 10 min, dehydrated in a graded series of ethanols (70% for 5 min, 80%

for 2 min, 95% for 2 min, 100% for 1 min), delipidated in 100% chloroform (5 min) and

partly rehydrated in 100% and 95% ethanol (1 min each), and allowed to dry.

For hybridization of the cryostat sections, labeled probes were diluted in a hybridization

buffer (10 ml labeled probe/ml hybridization buffer) consisting of 50% (v/v) formamide,

46SSC (150 mM NaCl, 15 mM sodium citrate, pH 7.0), 16Denhardt solution (0.02%

bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 10% (w/v) dextran

sulfate, 10 mM DDT, 0.5 mg/ml salmon sperm DNA, and 250 mg/ml yeast tRNA. An

aliquot of 200 ml of the hybridization buffer with the labeled probes (one oligonucleotide

probe for the b1-receptor and a mixture of the three oligonucleotide probes for the AA-NAT

enzyme) were pipetted onto sections. The sections were covered with parafilm and incubated

in a humid chamber overnight at 378C. After hybridization, the slides were washed in 16SSC

(4615 min at 558C and 2630 min at room temperature) and dipped twice in distilled water.

The sections were dried and either exposed to an X-ray film for 2 – 6 weeks in a light-tight

cassette or dipped in Amersham LM-1 emulsion at 408C and exposed for 4 – 8 weeks at 48Cin a light-tight slide box containing silica gel. The X-ray films were developed in a commercial

developing machine. The emulsion-coated slides were developed in Amidol for 5 min and

fixed in 30% thiosulfate.

Quantification of mRNA signal

The signals in the pineal were quantified by transferring the image of the sections to a

MacIntosh computer equipped with a capture board and running the program ‘‘Image 1.42’’

by Wayne Rasband, NIH, USA. The optical density of the autoradiographs was measured in

dpm/mg tissue by comparison with simultaneously exposed 15C-standards that had been

previously calibrated by comparison with 35S brain-paste standards.

Spectrofluorometrical analysis of serotonin

The spectrofluorometrical method of Snyder et al. (1965) was used. Measurement of

excitation (385 nm) and emission (495 nm) wavelenghts was done after treatment with

ninhydrin in a hot near-neutral solution. The fluorescence was measured in a spectro-

fluorimeter using 1 cm quartz cuvettes. Serotonin creatinine sulphate (5-hydrotryptamine

Sigma) was used as the standard. Results were expressed as ng/mg tissue.

Assay for arylalkylamine N-acetyltransferase activity

The AA-NAT activity was determined by the method descibed by Champney et al. (1984).

Pineal glands were homogenized in 1 ml of 0.05 M of ice-cold potassium phosphate buffer


(pH: 6.8). Twenty ml of each homogenate were mixed with 5 ml of 5.6 mM of serotonin

(Sigma, Chemical Co., St. Louis, MO, USA), 10 ml of 2.5 mmol acetyl-1-14C coenzyme A

(55.6 mCi/mmol, NEN Dupont Research Products, Boston MA, USA) and 10 ml of

phosphate buffer. The reaction was carried out at 378C for 10 min and stopped by

the addition of 100 ml of 0.5 M borate buffer (pH 10.0). The reaction mixture was

transferred into a test tube containing 1 ml of chloroform, and shaken for 3 min.

After centrifugation, 600 ml of the organic phase were transferred and evaporated to

dryness. The residue was dissolved with 1 ml of ethanol, 10 ml of scintillation solution was

added, and the radioactivity was measured. Results were expressed as pmoles/mg tissue/

10 min.

Assay for hydroxyindole-O-methyltransferase activity

Pineal HIOMT was assayed as previously described by Axelrod and Weissbach (1961).

Individual pineal glands were homogenized in 1 ml of 0.05 M sodium phosphate buffer (pH

7.9). 50 ml of the tissue homogenate were incubated for 30 min at 378C with 30 ml of 3 mM

N-acetylserotonin and 10 ml of 14 nmol/l S-adenosyl-L-[methyl-14C]methionine (57.6 mCi/

mmol, NEN Dupont Research Products, Boston MA, USA) in a final volume of 100 ml. After

incubation, the reaction was stopped by the addition of 0.3 ml of sodium borate buffer

(0.5 M, pH 10.0). Melatonin was extracted with 1 ml of water-saturated chloroform. The

organic phase was evaporated to dryness. The residue was dissolved with 1 ml of ethanol,

10 ml of scintillation solution was added, and the radioactivity was measured. HIOMT

activity is expressed as pmol/mg tissue/30min.

Pineal melatonin

The pineal glands were homogenized in 1 ml of ice-cold 0.05 M sodium borate buffer (pH:

10.0). The homogenates (1 ml) were mixed with 4 ml of methanol for 30 s and centrifuged

at 1500g at 48C for 10 min (Di et al. 1998). The supernatants were transferred to another

tube and dried in a rotary-vacuum evaporator under reduced pressure. The residues

were stored at 7208C until melatonin determination by radioimmunoassay could be carried

out.

The dried residues were reconstituted in 1 ml of 0.1 M tricine buffer (pH 5.5). Melatonin

estimates were done in duplicate using a radioimmunoassay as described by Fraser et al.

(1983) with a lower limit of sensitivity of 2.5 – 5.0 pg/tube.

Five hundred ml of each standard or sample in buffer tricine and 200 ml of a specific

antiserum to melatonin raised in sheep (Stockgrand Ltd.) were mixed and incubated at room

temperature for 30 min. Hundred ml of 3H-melatonin were added to all tubes and incubated

for 18 h at 48C. Antibody-bound melatonin was separated from the free fraction by

incubation for 15 min at 48C in ice with 500 ml dextran-coated charcoal. The tubes were spun

at 1500g for 15 min at 48C. Seven hundred ml aliquots of the supernatants were removed to

vials containing 3.0 ml scintillation fluid. Vials were shaken at room temperature for 1 h.

Radioactivity was counted in all tubes. The melatonin concentrations in the samples were

determined from a standard curve and expressed as pg/mg tissue.

Statistical analysis

Means and standard errors were calculated for all data sets. Differences between groups were

evaluated using One-way Analysis of Variance (ANOVA) or the Kruskal – Wallis Test


followed by the Tukey – Kramer or Dunn’s Multiple Comparisons Test, respectively. A

probability of less than 0.05 was assumed to be significant.

Results

In this study, we investigated daily rhythms of biochemical parameters involved in melatonin

synthesis in the viscacha pineal gland.

The norepinephrine content in the pineal gland showed significant daily variations, with

elevated levels at night-time compared with those at light-time (Figure 1). In fact, nore-

pinephrine was higher at ZT16 and ZT20. A diurnal lower increase at ZT4 and minimal

levels at ZT8 and ZT12 were observed.

A daily variation in the mRNA encoding b1-adrenoceptor was observed by using

in situ hybridization (Figure 2). A quantitative analysis of pineal sections showed that

b1-adrenoceptor mRNA expression peaked at ZT8, followed by a decrease, reaching the

minimun at ZT0.

Significant daily differences in serotonin concentrations were also observed with the highest

value occuring at day-time (ZT4) in comparison with those at night-time (ZT16 and ZT20)

(Figure 3).

In situ hybridization studies for mRNA encoding the arylalkylamine N-acetyltransferase also

revealed a daily rhythm in the viscacha pineal gland with the highest expression at ZT0. The

levels decreased significantly at ZT4 reaching the minimum at night, ZT16 and ZT20

(Figure 4).

Likewise, the daily activity of the arylalkylamine N-acetyltransferase enzyme is shown in

Figure 5. The AA-NAT activity in the viscacha pineal gland exhibited a pronounced and

significant nocturnal peak at ZT16, followed by a significant decrease in the enzymatic

activity at ZT20 and a lower diurnal increase at ZT0 and ZT4, reaching a minimun at ZT8.

Figure 1. Diurnal variation in the pineal norepinephrine. The values are means+S.E.M from four animals. The NE

levels show statistically significant differences as follows: One-way ANOVA: p5 0.0001, followed by Tukey’s

Multiple Comparison Test: ZT20 versus ZT0, ZT8 and ZT12; ZT4 versus ZT12; ZT8 versus ZT16; ZT12 versus

ZT16 p50.001; ZT20 versus ZT4; ZT0 versus ZT12 and ZT16; ZT4 versus ZT8 p50.01; ZT20 versus ZT16;

ZT0 versus ZT8 p50.05.


On the other hand, the HIOMT activity in the viscacha pineal gland was also rhythmic

(Figure 6), with a significant peak at ZT16. Enzymatic activity was constantly lower in the rest

of the day.

Finally, the daily melatonin content in the viscacha pineal gland was determined by using

radioimmunoassay (Figure 7). The pineal melatonin showed a daily rhythm characterized by

a short and pronunced increase at ZT16. This peak was followed by a drastic decrease at

ZT20. The indole levels in the gland increased significantly during the light period, ZT0 and

ZT4, reaching the minimun at ZT8.

Figure 3. Daily rhythm of pineal serotonin. Values are means+S.E.M. from four animals. The serotonin levels show

statistical differences: One-way ANOVA p5 0.0001, followed by Tukey’s Multiple Comparison Test: ZT4 versus

ZT8, ZT12, ZT16, ZT20, ZT0 p5 0.001, ZT0 versus ZT16; ZT8 versus ZT16 p5 0.01.

Figure 2. The diurnal expression of b1-adrenoceptor mRNA hybridization signal. Each bar represents mean+S.E.M.

from four animals. Statistically analysis was performed by Kruskal – Wallis Test p5 0.0001, followed by Dunn’s

Test: ZT8 versus ZT20, ZT0, ZT4, ZT12, ZT16 p5 0.001, ZT0 versus ZT4, ZT16 p50.01, ZT0 versus ZT12

p50.05.


Discussion

Melatonin, the major hormone produced by the pineal gland, displays charactheristic daily

and seasonal patterns of secretion, and plays an important role in the timing of many

physiological functions.

The retinohypothalamic – pineal (RHP) axis is capable of detecting changes in the length of

the night and making proportional adjustments in the duration of nocturnal melatonin

secretion from the pineal gland (Goldman 1983; Bittman & Karsch 1984; Hoffmann 1985;

Karsch et al. 1991; Wehr 1996, 1997). The pineal gland is a key component of the RHP axis,

Figure 4. Diurnal variation in the expression of mRNA encoding AA-NAT enzyme. Values are means+S.E.M. from

four animals. There are significant differences (Kruskal – Wallis test: p5 0.0001, followed by Dunn’s test: ZT20

versus ZT0, ZT4, ZT8; ZT0 versus ZT8, ZT12, ZT16; ZT4 versus ZT12, ZT16 p5 0.001, ZT4 versus ZT8 and

ZT8 versus ZT16 p50.01, ZT0 versus ZT4 and ZT8 versus ZT12 p5 0.05).

Figure 5. Daily rhythm of AA-NAT activity. The values are the means+S.E.M. from four animals. Statistical analysis

was performed by Kruskal – Wallis (p5 0.006), and followed by Dunn’s test: ZT8 versus ZT16 p5 0.01.


AA-NAT and HIOMT being two enzymes involved in rhythmic synthesis of melatonin.

Melatonin daily rhythms are due primarily to changes in the AA-NAT activity

(Axelrod 1974; Klein et al. 1981, 1996; Roseboom et al. 1996; Cahill & Besharse 1995).

Recent studies suggest that HIOMT plays an important physiological role in the regulation of

melatonin production (Ribelayga et al. 2000; Simonneaux & Ribelayga 2003). There is

evidence that melatonin synthesis is regulated by postganglionic sympathetic fibers

and their neurotransmitter norepinephrine. Ceinos et al. (2004) demonstrated that this

regulation is more complex than an only adrenergic control of AA-NAT expression and

activity.

Figure 6. Daily rhythm of HIOMT activity. The values are the means+S.E.M. from four animals. There are

significant differences (ANOVA one-way p5 0.002, followed by Tukey’s test: ZT16 versus ZT20, ZT0, ZT4 y ZT8

p50.01 y ZT16 versus ZT12 p5 0.05).

Figure 7. Daily rhythm of melatonin levels. The values are means+S.E.M. from four animals. Statistical analysis was

performed by one-way ANOVA (p50.001) and followed by Tukey’s test: ZT16 versus ZT8 p50.001, ZT16 versus

ZT20, ZT16 versus ZT12 p50.01, ZT0 versus ZT8, ZT4 versus ZT8 p5 0.05).


Previous studies performed by our group revealed that the adult male viscacha is a seasonal

breeder which exhibits daily rhythms of melatonin in pineal, serum and retina (Fuentes et al.

1991, 1993, 2003, 2004; Domınguez et al. 1987; Pelzer et al. 1999; Munoz et al. 1997, 1998,

1999, 2001; Calderon et al. 2001). In the wild this nocturnal rodent would only perceive light

signals at dawn and at dusk. Therefore, even though the photoperiod represents a key

stimulus in its breeding behavior; food and water availability and temperature might facilitate

it. Together this evidence makes this wild rodent an interesting model to elucidate the

complex mechanisms involved in the control of daily cycle activity, which have not been

completely elucidated.

The night-time production of melatonin is mainly driven by the circadian clock, situated in

the suprachiasmatic nucleus of the hypothalamus, which controls the release of NE from the

dense pineal sympathetic afferents fibers. The role of NE in the nocturnal stimulation of

melatonin synthesis has been extensively dissected at the cellular and molecular levels (Ceinos

et al. 2004; Drijfhout et al. 1996; Simonneaux & Ribelayga 2003). In this study, we show that

NE content in the viscacha pineal gland is rhythmic with the highest levels at night. Levels

decreased during the light period, being at minimum in the early night. Therefore, we argue

that the daily NE pattern in viscacha might be mainly driven by the day/night cycle; however,

other Zeitgebers might be responsible for the diurnal increase.

A daily rhythm in the mRNA encoding the b1-adrenergic receptor, with maximal levels

during the light phase, was observed in the viscacha pineal gland. This pattern is similar to

that observed in the Syrian hamster pineal b-adrenergic receptor density with high values in

the light phase (Pangerl et al. 1990). This significant increase in the levels suggested a

significant upregulation during the light-phase. In addition, the photoperiodic information

would regulate the expression of the b1-adrenoceptor gene throughout NE stimulation. The

mechanisms underlying this mode could be related to other controls’ cellular and molecular

processes. Studies in the rat pineal gland indicate that the levels of the b1-adrenergic receptor

mRNA are regulated by the sympathetic mechanism transmitted through superior cervical

ganglia and also regulated by the inhibitor factors from the cyclic AMP-signaling pathway in a

feedback mechanism (Pfeffer et al. 1998).

Beside the rhythmic melatonin secretion, another consequence of the diurnal changes in

AA-NAT activity is a prominent rhythm of the pineal serotonin content. The performed

studies in viscacha demonstrated the presence of a diurnal rhythm in the pineal serotonin

content with the highest level in the middle of the light phase and the lowest in the dark phase.

Therefore, serotonin depletion at night is a consequence of its convertion into melatonin

through AA-NAT. In contrast, the activity of this enzyme showed a prominent diurnal rhythm

with the highest level during the night and low levels during the day. These data could indicate

that daily variation of pineal indoles may not only be dependent on changes in the AA-NAT

activity, but that other mechanisms regulating pinealocyte serotonin availability could also be

involved, such as was demonstrated in Djungarian hamster (Miguez et al. 1996).

On the other hand, mRNA encoding AA-NAT in the viscacha pineal gland exhibited a

daily pattern characterized by a pronouced increase during the early day, and after that

the expression decreased significantly. The general pattern of expression seen in all the

vertebrates examined is that AA-NAT mRNA is always strongly expressed at night in the

pineal gland (Coon et al. 1995). However, in viscacha, a correlation between the b1

adrenoceptor and AA-NAT mRNA has not been observed, which suggests that other

mechanisms not related directly to b1-adrenoceptor activation and cAMP might be involved

in the expression of the AA-NAT gene.

Furthermore, previous studies have revealed that the relationship between mRNA and AA-

NAT activity vary among species. These observations indicate that transcriptional and/or


post-transcriptional mechanisms may regulate AA-NAT activity involved in the different

species (Klein et al. 1997). In viscacha, the pattern observed in AA-NAT activity of the pineal

gland showed a strong and significant peak at night and was followed by a significant decrease

in the enzymatic activity of the gland. The temporal patterns of AA-NAT mRNA and enzyme

activity were found not to be similar in the viscacha pineal gland. These data suggest that post-

transcriptional mechanisms might play a role in the regulation of this enzyme. Several possible

mechanisms might generate the rhythmic changes in AA-NAT mRNA, such as translocation

or association of stable proteins that regulate the abundance of AA-NAT mRNA by

controlling either its transcription or its degradation rate, or rhythmic post-translational

modifications of pre-existing transcription factors (Schomerus et al. 2000; Choi et al. 2004).

Previous studies demonstrate that at least part if not all the phase-shifting/entraining effects of

light on the AA-NAT activity rhythm occur at the mRNA level, assuming that light is acting

on AA-NAT mRNA primarily through effects on the clock (Bernard et al. 1997). However,

other studies demonstrated that AA-NAT mRNA is constitutively high throughout the 24 h-

cycle, and melatonin production is primarily controlled through effects on AA-NAT enzyme

activity, in contrast to dominant transciptional control in rodents (Johnston et al. 2004).

Continued analysis of this system should provide further insight into how the clock controls

the rhythm in AA-NAT mRNA and how it regulates AA-NAT activity in the Lagostomus

maximus maximus.

The present study reveals a unique diurnal rhythm in viscacha pineal HIOMT. The peak

in enzyme activity occurs 5 h after the onset of darkness, followed by a significant decrease

to being level and constantly stable during the rest of the day. The peak in HIOMT activity

is consistent with that in melatonin content, suggesting HIOMT participation in the

indole increase. Sugden and Klein (1983) suggested that the neural control of pineal

HIOMT is mediated via a beta adrenoceptor. The approximately two-fold nocturnal

increase in the methylating capacity in the viscacha pineal gland may be attributed to NE

stimulation.

During the period of maximal testicular activity, the melatonin profile in the viscacha pineal

gland is characterized by a nocturnal peak similar to that decribed in the Syrian hamster and a

second diurnal increase (Calderon et al. 2001). In this period of the reproductive cycle,

melatonin peak was short and pronounced during darkness. This peak was followed by a

drastic decrease. The indole levels in the gland increased significantly during the light period

reaching a minimum in the afternoon.

It is well known that in photoperiodic mammals studied to date, melatonin plays an

important role adjusting to physiological processes. In viscacha, the nocturnal stimulation of

melatonin synthesis in the pineal gland might be mainly driven by the nocturnal release of

NE. Daily changes in AA-NAT activity are coincident with those of melatonin production,

and the nocturnal increase of melatonin is similar to the peak of activity of HIOMT.

Daily variations in HIOMT activity suggest an important contribution of HIOMT to the

melatonin rhythm in this photoperiodic species. We speculate that the nocturnal increase of

AA-NAT and HIOMT activities results from adrenergic stimulation. In addition, we may not

exclude that some neuropeptides present in the pineal gland may also be involved in this

process.

The comparison of the results obtained in this rodent and those previously investigated in

other mammals suggests large interspecies differences in the diurnal changes occurring in the

pineal gland. These interspecies differences probably reflect a considerable heterogeneity in

the ultrastructure (Bhatnagar 1992; Lewczuk et al. 2004) and in the innervation of the pineal

gland (Nowicki et al. 2003; Przybylska-Gornowickz et al. 2000) as well as, in the mechanisms

governing melatonin synthesis (Ganguly et al. 2002; Lewczuk 2002).


Our data suggest that the differences found among the viscacha and terrestrial ruminants

and fossorial rodents is likely to be related to the lifestyle or be consistent with evolutionary

divergence in the mechanisms regulating melatonin production.

In conclusion, our results, in conjunction with previous observations, demonstrate that the

adrenergic system might be involved in the viscacha daily cycle, the photoperiod being an

important environmental signal capable of triggering changes in its behavior and physiology.

In addition, new studies are necessary to elucidate the chemical signals, target sites, and

mechanisms related to the day/night regulation and/or modulation of melatonin synthesis in

the viscacha pineal gland.

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