NEL, Melatonin: Clinical Significance and Therapeutic Application

M. Pawlikowski, M. Kolomecka, A. Wojtczak & M. Karasek:
Effects of six months melatonin treatment on sleep quality and "serum concentrations of estradiol, cortisol, dehydroepinadrosteron sulfate, "and somatomedin C in elderly women
2002; 23 (suppl 1):17-19 pii:NEL230702A01 PMID:

Submitted: October 10, 2001 Accepted: October 31, 2001

Key words:
melatonin; estradiol; DHEAS; cortisol; IGF-I; aging

Abstract
OBJECTIVES: The role of melatonin is aging is still under debate. Therefore, an open pilot study on the effects of melatonin administration on some sleep parameters, routine hematological and biochemical parameters, and concentrations of hormones was performed in elderly women.

SUBJECTS AND METHODS: The study was performed on 14 women (volunteers), aged from 64 to 80 years (mean age 71±4.6 years). Melatonin (2 mg daily at 19:00 h) was administered during 6 months. Before and after melatonin treatment the peripheral venous blood samples were taken in the morning (approx. at 08:00 h) after the overnight fast. The total blood count, glucose, total cholesterol, LDL, HDL, and triglycerides were estimated by routine laboratory methods. The serum concentrations of the following hormones were determined: 17-beta-estradiol, dehydroepiandrosterone sulfate (DHEAS), cortisol, and somatomedin C (IGF-I). Additionally, before and after 6 months of melatonin therapy the investigated subjects answered to a questionnaire dealing with sleep parameters and self-estimation of general health status.

RESULTS: In 35.7% of investigated subjects an improvement in general sleep quality and in such sleep parameters as sleep initiation, sleep latency, number of awakenings episodes, wake time after sleep onset, was observed. A significant decrease of estradiol concentrations was observed after 6 months of the melatonin treatment in comparison to initial levels. IGF-I was found to be slightly but significantly increased after the 6 months melatonin therapy. Cortisol levels did not change significantly, during the melatonin treatment. DHEAS concentrations increased after melatonin therapy. Moreover, a tendency towards a higher DHEAS/cortisol ratio was found after 6 months of treatment. Melatonin treatment did not influence significantly either the parameters of total blood count or glucose and serum lipids levels.

CONCLUSIONS: On the basis of this preliminary open study it seems that melatonin administration may be beneficial for elderly subjects.

Introduction
Although the role of melatonin in the aging process is still under debate, there are some data suggesting that melatonin administration may be beneficial in advanced age [1], especially in elderly insomniacs [2, 3]. Melatonin secretion exhibit a clear circadian rhythm with low values during the daytime and 8-15 fold increase at night [1, 4, 5]. This nocturnal peak in melatonin levels decrease significantly with age [1, 5]. Recently melatonin received a great deal of attention as therapeutic agent in various diseases. Moreover, safety of melatonin treatment is discussed [6]. Because the data on melatonin use in advanced age are scarce we decided to perform an open pilot study on the effects of melatonin administration on some sleep parameters, routine hematological and biochemical parameters, and concentrations of hormones in elderly women.
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A. Rimler, Z. Lupowitz & N. Zisapel:
Differential regulation by melatonin of cell growth and androgen receptor "binding to the androgen response element in prostate cancer cells
2002; 23 (suppl 1):45-49 pii:NEL230702A02 PMID:

Abstract
OBJECTIVES: The pineal hormone melatonin inhibits the growth of benign human prostate epithelial cells and the androgen-dependent prostate cancer LNCaP cells. In the androgen-nonresponsive prostate carcinoma PC3 cells melatonin inhibits cell growth only at high but not low cell density. We have recently found that melatonin causes nuclear exclusion of the AR and attenuates it transcriptional activity in LNCaP cells as well as PC3 cells stably transfected with a wild type AR expressing vector (PC3-AR). The aim of this study was to investigate whether melatonin inhibits effects of AR on cell growth in PC3-AR cells and whether inhibition of AR DNA binding is involved.

METHODS: The effects of androgen, melatonin and their combination on the growth of the PC3-AR cells and on AR DNA binding in PC3-AR and LNCaP cells were studied.

RESULTS: DHT suppressed cell growth in the PC3-AR cells and enhanced AR binding to the androgen responsive element (ARE). Melatonin had no effect on cell growth in the absence of DHT but counteracted the androgen-induced inhibition at low androgen concentrations. Melatonin did not suppress and even slightly enhanced the capacity of AR binding to the ARE in the PC3-AR as well as in LNCaP cells.

CONCLUSIONS: Attenuation by melatonin of AR activity in the prostate cancer cells is not due to suppression of AR binding to the ARE, and is presumably caused by its effects on AR protein interaction and intracellular trafficking.

Introduction
Melatonin, the hormone secreted at night by the pineal gland, inhibits the growth of human benign (BPH) and tumor (LNCaP) prostate epithelial cells in vitro [1-4]. Melatonin inhibits growth of the prostatic carcinoma PC3 cells which do not express the androgen receptor (AR) only at high cell density and slightly enhances growth at low cell density [5]. The androgen receptor (AR) is an intracellular receptor that functions as a ligand dependent transcription factor [6]. The AR mediates all the activities ascribed to androgens, including development, differentiation and maintenance of male reproductive function, support of sexually dimorphic non-reproductive functions and prostate cancer growth. In androgen target tissues, the AR is localized within the nucleus [7]. Upon ligand binding, the AR binds to hormone response elements in the promoter region of inducible genes thus controlling their transcription [6]. Mutations at the DNA binding domain of the AR, leading to nuclear exclusion of the receptor and loss of androgen sensitivity, have recently been identified in prostate cancer cells [8].
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K. Winczyk, M. Pawlikowski, H. Lawnicka, J. Kunert-Radek, G. Spadoni,
G. Tarzia & M. Karasek:
Effect of melatonin and melatonin receptors ligand
N-[(4-methoxy-1H-2-yl)methyl]propamide on murine Colon 38 cancer growth in vitro and in vivo
2002; 23 (suppl 1):50-54 pii:NEL230702A03 PMID:

Key words:
melatonin; melatonin receptors; proliferation; apoptosis; colon cancer

Abstract
OBJECTIVES: Our previous study suggest that oncostatic action of melatonin (MLT) depends mainly on nuclear RZR/ROR receptors. However, we cannot exclude the involvement of membrane receptors in the control of tumor growth. In the present study the effects of MLT and N-[(4-methoxy-1H-indol-2-yl)methyl]propanamide (UCM 386 - antagonist of membrane MT1 receptor and partial agonist of membrane MT2 receptor) on murine transplantable Colon 38 cancer were investigated in vitro and in vivo conditions.

MATERIAL AND METHODS: The experiments were performed on adult male B6D2F1 mice strain. In vitro the cell proliferation was measured using modified Mosmann method. In the experiment performed in vivo, we assessed the cell proliferation, apoptosis and proliferation/apoptosis ratio (P/A). The incorporation of bromodeoxyuridine into tumor cell nuclei was used as an index of cell proliferation (labeling index-LI). The labeling of apoptotic cells according to TUNEL method was considered as an index of apoptosis (AI).

RESULTS: In vitro MLT and UCM 386 decreased the cell proliferation, but administration of MLT and UCM 386 together did not change the inhibitory effect of MLT alone. In vivo MLT and UCM 386 alone decreased LI and the addition of UCM 386 to MLT did not diminish the antiproliferative effect of MLT. Melatonin and UCM 386 injected alone also increased the AI. Moreover, both compounds given together exerted the additive effect on tumor apoptosis. MLT and UCM 386 alone or together also significantly decreased P/A ratio which is additional parameter confirming the inhibition of tumor growth.

CONCLUSION: The obtained data together with our earlier observations suggest that oncostatic effect of MLT depends on acting via both MT2 and RZR/ROR nuclear receptors

Melatonin (MLT), the hormone of pineal gland, is a new candidate as anticancer agent. Recently, the oncostatic action of melatonin on various experimental cancers has been reported. The antitumor effect of melatonin is connected, in part with antiproliferative and also proapoptotic activities. The number of investigations confirmed the antiproliferative action of melatonin [1, 2, 3]. The previous study conducted in our laboratory on murine Colon 38 cancer shows that MLT causes not only the inhibition of tumor cell proliferation, but also induces apoptosis [4]. The mechanism by which MLT exerts its antitumor effect is very complex and still not clear. This pineal hormone may act indirectly via modulation of endocrine and immune systems. Melatonin may also influence directly on tumor cells through the specific binding sites. The best known melatonin binding sites are two subtypes of receptors, named MT1 and MT2 [5]. It was also suggested that MLT is a natural ligand for the nuclear orphan receptors RZR/ROR [6, 7]. Moreover, it was proposed that immunological and oncostatic effects of melatonin depend mainly on nuclear signaling [8, 9]. ... ...

Key words:
melatonin receptors; MT1 receptor; MT2 receptor; RZR/RORa receptor; colon cancer

Abstract
OBJECTIVES: There are some data suggesting that melatonin exerts oncostatic action through membrane as well as nuclear receptors. In previous studies we demonstrated the antiproliferative and proapoptotic action of melatonin on transplantable murine Colon 38 adenocarcinoma cells. Therefore, the aim of the present study was to determine whether the membrane melatonin receptors MT1 and MT2 as well as the nuclear receptor RZR/RORa are expressed in Colon 38 cells.

MATERIAL AND METHODS: Adult male B6D2F1 mice were used in this experiment. The induction of tumor was conducted by subcutaneous injection of 0.2 mL of a 33% suspension of Colon 38 cancer cells into axillary region. Expression of mRNA encoding MT1 and MT2 melatonin membrane receptors was studied by RT-PCR analysis, and expression of RORa1 nuclear receptor protein was studied by Western blot analysis.

RESULTS: The expression of mRNA encoding both MT1 and MT2 melatonin receptors was demonstrated in Colon 38 cancer cells. Moreover, immunodetection revealed the expression of MT1 and RORa1 proteins in these cells.

CONCLUSIONS: Our studies on Colon 38 adenocarcinoma cells support the concept that both membrane and nuclear receptors are involved in the oncostatic action of melatonin.

There is substantial experimental evidence indicating inhibitory influence of pineal hormone melatonin on the malignant tumor formation and/or growth [1-4]. In the previous studies we demonstrated that in transplantable murine Colon 38 adenocarcinoma melatonin exerts inhibitory effect on the tumor cell proliferation and stimulatory effect on tumor cell apoptosis [5-9]. Moreover, we suggested that melatonin decreases the tumor proliferation acting through both membrane and nuclear receptors. On the other hand, the induction of apoptosis by melatonin seems to depend mainly on its action through RZR/RORa receptor [8, 9]. Therefore, the aim of the present study was to determine whether membrane melatonin receptors MT1 and MT2 as well as nuclear receptor RZR/RORa are expressed in Colon 38 cells.
... ...

M. Markowska, A. Mrozkowiak & K. Skwarlo-Sonta:
Influence of melatonin on chicken lymphocyte in vitro:
involvement of membrane receptors
2002; 23 (suppl 1):67-72 pii:NEL230702A05 PMID:

Key words:
melatonin; signal transduction; proliferation; camp; luzindole; chicken; splenocytes

Abstract
OBJECTIVES: Time-dependent melatonin effects on chicken lymphocyte proliferation in vitro and the involvement of cAMP in melatonin signal transduction were examined.

MATERIALS AND METHODS: Splenocytes and peripheral blood mononuclear cells (PBMC) were cultured in vitro in the presence of melatonin, phytohemagglutinin, luzindole, dibutyrylcAMP (dbcAMP), forskolin and vasoactive intestine peptide (VIP). Proliferation was measured by [3H]-thymidine incorporation in cultures carried out for 24, 36, 48 and 72 h. Cyclic AMP formation was assessed by radioimmunoassay in cells incubated for 30 min. or 24 h.

RESULTS: Melatonin stimulated the spontaneous proliferation in short-term (36 and 48 h) splenocyte cultures and had no effect in 72 h cultures. It inhibited mitogen-stimulated proliferation already in 24 h cultures and this effect was observed regardless of the time of the culture. Both melatonin effects were antagonized by luzindole - membrane-bound melatonin receptor antagonist. Forskolin and dbcAMP caused a significant inhibition of proliferation of splenocytes and PBMC cultured for 24 or 72 h, respectively. Melatonin inhibited the cAMP formation (30 min. of incubation) stimulated by adenosine cyclase activators - forskolin and VIP, but added alone failed to affect the cAMP concentration. In mitogen-stimulated splenocytes cultured for 24 h Mel caused an increase in cAMP correlated with the inhibition of cell proliferation.

CONCLUSIONS: Melatonin effects on chicken splenocytes appears time- and activation-dependent: in short-term cultures it stimulates spontaneous and inhibits mitogen-activated proliferation, probably via membrane-bound, luzindole-sensitive melatonin receptors. Incubation with melatonin for 30 min. inhibits cAMP formation, but in 24 h cultures it increases cAMP concentration leading to inhibition of proliferation.

A. Bortkiewicz, B. Pilacik, E. Gadzicka & W. Szymczak:
The excretion of 6-hydroxymelatonin sulfate in healthy young men exposed to "electromagnetic fields emitted by cellular phone - an experimental study
2002; 23 (suppl 1) 88-91 pii:NEL230702A06 PMID:

Key words:
melatonin; 6-hydroxymelatonin sulfate; magnetic field; cellular phone

Abstract
OBJECTIVES: It is quite likely that non-visible electromagnetic fields (EMF) may affect melatonin production. Some studies confirmed this hypothesis and showed that extremely low EMF altered pineal function in animals and humans. Thus, it is reasonable to suppose that EMF emitted by cellular phones may also influence secretion of melatonin. The present study sought to evaluate possible effect of the exposure to EMF emitted by cellular phone on 6-hydroxymelatonin sulfate (6-OHMS) excretion, which reflects melatonin levels in blood.

MATERIAL AND METHODS: The examined group consisted of 9 healthy males aged 19-29 years. The experiment was performed under controlled conditions (the light intensity-50 lx till midnight and 0 lx during night). Each person was examined twice: on a day without exposure (control day, C-day) and on a day with continuous exposure (60 min. exposure from cellular phone, frequency 900 MHz, pulsed with 217 Hz, pulse with 576 µs, SAR 1.23 W/kg, E-day). From 7 p.m. to 8 p.m. they used a cellular phone. The subjects did not know which day was E-day, and which was C-day. From 8 p.m. till midnight the subjects listened to music and than they slept till 7 a.m. next day. Urine samples were collected at 7 p.m., at midnight, and at 7 a.m. in the same way in C-day as in E-day. Sample were frozen for later ELISA analysis of 6-OHMS. The 6-OHMS ELISA kit from Immuno-Biological Laboratories (Hamburg) was used for measurement of 6-OHMS. The data were analysed using Wilcoxon matched-pairs signed-ranks test for each subject and for the whole group. We compared 6-OHMS level on the E-day and on the C-day separately for 3 time-points - 7 p.m., midnight, 7 a.m.

RESULTS: Mean 6-OHMS level in both experiments did not differ significantly for any of the respective time points. Circadian variations of 6-OHMS level were detected in all subjects.

CONCLUSIONS: The results of our investigation has demonstrated that EMF emitted by cellular phones has no distinct influence on the melatonin level.

The increasing use of mobile phones has caused growing interest in possible health effects of electromagnetic fields which they produce. Although exposure caused by cellular phones does not exceed the admissible levels, it is worth noting that the standards were developed on the basis of expected thermal effects and do not consider possible effects of chronic, non-thermal exposures. Studies on this subject are still sparse and incomplete, some of them report disturbances in various physiological functions associated with such exposure; non-specific neurovegetative disorders, headaches, muscle pains, sleep disturbances, and increased arterial blood pressure have been observed in the exposed subjects [1-5]. A lot of physiological and biochemical functions are influenced by melatonin. Considering the significance of the effects of visible light on the pineal function, theoretically it is quite likely that non-visible electromagnetic fields may affect melatonin production. Some studies confirmed this hypothesis and showed that extremely low electromagnetic fields altered pineal function in animals and humans [6]. Thus, it is reasonable to suppose that radio frequency electromagnetic fields emitted by cellular phones may also influence pineal production and secretion of melatonin. However, either the experimental investigations on animals nor on humans have brought a conclusive answer to this question. The objective of this study was to determine whether 1-hour exposure to electromagnetic fields emitted by cellular phone suppressed nocturnal melatonin production. The present study sought to evaluate possible effects of the exposure on 6-hydroxymelatonin sulfate (6-OHMS) excretion, which reflects melatonin levels in blood.
... ...

M. Karasek, A. Szuflet, W. Chrzanowski, K. Zylinska & J. Swietoslawski:
Circadian serum melatonin profiles in patients suffering from chronic renal failure
2002; 23 (suppl 1):97-102 pii:NEL230702A07 PMID:

Key words:
melatonin; chronic renal failure; end-stage renal disease;
circadian rhythm

Abstract
OBJECTIVES. In spite of broad interest, intensive studies on function of melatonin have not yielded much information about relationships between this hormone and kidneys in health, and particularity, in disease. Very little is known about the circadian plasma melatonin concentrations in patients with chronic renal failure (CRF). There are only a few studies dealing with melatonin concentrations in renal diseases, mainly performed in hemodialyzed patients with end-stage renal disease (ESRD). Moreover, the most melatonin assays were performed during the daytime, and the results are conflicting. Therefore, the aim of the present study was to determine the circadian melatonin profiles in patients with different stages of CRF.

MATERIAL AND METHODS. Twenty four patients (13 males and 11 females) with CRF aged 35 to 58 years (mean±SEM: 47.0±1.6 years) were included in the study. Patients were divided into two groups: group 1 - patients with compensated CRF (serum creatinine: 2.0-5.0 mg/dL), group 2 - patients with ESRD (serum creatinine: > 8,0 mg/dL). The control group consisted of 20 healthy volunteers (10 males and 10 females) aged 35 to 55 years (mean±SEM: 46.0±1.5 years) checked not to have renal failure [serum creatinine: 0.8-1.4 mg/dL], and matched according to sex and age. Blood samples were collected at 08:00, 12:00, 16:00, 20:00, 24:00, 02:00, 04:00, and 08:00 h. Melatonin concentration was measured by enzyme immunoassay.

RESULTS. In both groups of patients with chronic renal failure, i.e. in patients with compensated disease and in patients with end-stage renal disease melatonin nocturnal concentrations were significantly lower then those in healthy volunteers. Moreover, in patients with compensated renal failure also day-time melatonin concentrations were significantly depressed. Area under curve was significantly lower in both groups of patients in comparison with the control group.

CONCLUSIONS. The mechanism of depressed melatonin concentrations in CRF observed in our study remains unclear. However, it seems possible that decline in melatonin levels is due to impairment in adrenergic function that occurs in CRF. Because the studies on the melatonin secretion in CRF bring about conflicting results, the relationship between renal diseases and melatonin secretion needs further investigations.

Melatonin is a major secretory product of the pineal gland. This hormone received recently great deal of interest due to its diversified action, including regulation of biological rhythms [1, 2], free radicals scavenging [3, 4], modulation of the immune system [5, 6], influence on neoplastic disease [7-9], and possible role in aging process [10, 11]. Although the abolished melatonin circadian rhythm and amplitude have been demonstrated in various diseases [12, 13], its precise role in different pathologies is still unknown.
Melatonin secretion exhibits typical circadian rhythm with low concentrations during the daytime (10-20 pg/mL) and high concentrations at night (70-120 pg/mL). The hormone is metabolized primarily in the liver and secondarily in the kidney. It undergoes 6-hydroxylation to 6-hydroxymelatonin, followed by sulfate or glucuronide conjugation to 6-hydroxymelatonin sulfate (90-95%) or 6-hydroxymelatonin glucuronide (5-10%). Melatonin also forms some minor metabolites. The main melatonin metabolite, 6-hydroxymelatonin sulfate is excreted in urine [1, 13].
Melatonin synthesis may be influenced by drugs. It is strongly reduced by b-blockers, whereas benzodiazepines, calcium antagonists, dexamethazone, nonsteroidal anti-inflammatory drugs, and clonidine are somewhat less active in this respect. Anti-depressive drugs stimulate melatonin secretion [1, 14].
In spite of broad interest, intensive studies on function of this hormone have not yielded much information about relationships between melatonin and kidneys in health, and particularity in disease. Very little is known about the circadian plasma melatonin concentrations in patients with chronic renal failure (CRF). There are only a few studies dealing with melatonin concentrations in renal diseases, mainly performed in hemodialyzed patients with end-stage renal disease (ESRD). Moreover, the most melatonin assays were performed during the daytime, and the results are conflicting [15-17].
Therefore, the aim of the present study was to determine the circadian melatonin profiles in patients with different stages of CRF.
... ...

Z. Ostrowska, B. Kos-Kudla, B. Marek, D. Kajdaniuk, P. Staszewicz,
B. Szapska & J. Strzelczyk:
The influence of pinealectomy and melatonin administration on "the dynamic pattern of biochemical markers of bone metabolism in experimental osteoporosis in the rat
2002; 23 (suppl 1):104-109 pii:NEL230702A08 PMID:

Key words:
pinealectomy; melatonin; bone metabolism; experimental osteoporosis; female rats

Abstract
BACKGROUND AND OBJECTIVES: There have been suggestions in literature that characteristic changes of bone mass in osteoporosis may be related to the melatonin (Mel): The aim of this study was to demonstrate whether pinealectomy and Mel administration can affect postmenopausal osteoporosis processes induced in female rats by way of ovariectomy.

METHODS: The study included 198 animals; 6 remained intact (0), 96 were ovariectomized (Ox), and the remaining ones underwent a sham operation (SOx). Two weeks after surgery, the rats were divided into eight groups: 1) SOx + SPx, 2) SOx + SPx + Mel, 3) Ox + SPx, 4) Ox + SPx + Mel, 5) SOx + Px, 6) SOx + Px + Mel, 7) Ox + Px, 8) Ox + Px + Mel. Animals from the 5th, 6th, 7th and 8th groups were pinealectomized (Px) while the remaining ones underwent a sham operation (SPx). Two weeks after surgery Mel (50mg/100g of bm) were administered intraperitoneally in rats in the 2nd, 4th, 6th and 8th groups while the remaining animals were administered with solvent only (5% solution of ethyl alcohol in physiological saline). Rats were administered the Mel solution or the solvent daily between 5 and 6 pm during a 4-week period. At the appropriate time, i.e. prior to surgery (group 0) and after 6, 12, 18 and 24 weeks from Px or SPx (time subgroups a, b, c and d) the animals were placed separately in metabolic cages (from 6.30 until 9.30 am) in order to collect urine aliquots for HYP and Ca determinations. The blood for the assay of ALP, PICP and ICTP was collected within the next 24 hours at 8 am (rats killed by decapitation).

RESULTS: The study has shown that pinealectomy had inducing, while exogenous Mel suppressing effect upon the level of investigated markers of bone metabolism; these changes were more pronounced in ovariectomized rats. Administration of Mel only partially levelled changes of bone metabolism caused by pinealectomy. In rats with preserved pineal gland effect of Mel on bone turnover markers was less pronounced. After discontinuing administration of Mel distinct tendency to increase studied biochemical markers of bone metabolism was shown.

CONCLUSION: Our findings suggest that Mel is an important modulator of experimental osteoporosis processes induced in female rats by way of ovariectomy.

Introduction
A few studies, particularly experimental, suggest that the main neurohormone of pineal gland - melatonin (Mel) can influence bone tissue metabolism. Our own studies performed previously on starved male rats have shown a significant suppressive effect of Mel on processes of bone formation and resorption [1]. Furthermore, it has been found [unpublished data] that illumination conditions, pinealectomy, and long-term Mel administration influence the circadian metabolism of bone tissue in male rats, and the changes in endogenous Mel concentrations seem to play an important role in the mechanism of this dependence. In an experimental model of postmenopausal osteoporosis, generated by ovariectomy in female rats, a successive lowering of nocturnal Mel concentration was observed [2]. These changes correlated, in an inversely proportional manner, with the values of biochemical markers of bone metabolism, particularly markers of bone resorption. As a consequence of these studies it seems that Mel deficiency, occurring probably as a result of switching off female sex hormone function, may be a cofactor in inducing bone mass changes in female rats with removed ovaries [2].
Clinical studies suggest that characteristic changes of bone mass in postmenopausal osteoporosis, resulting from predominance of resorptive processes over those leading to bone tissue formation, may be related to the Mel [3]. It has been suggested that pineal Mel is an anti-aging hormone [2-6] and that the menopause is associated with a substantial decline in Mel secretion and an increased rate of pineal calcification [8-10]. ... ...

B. Lewczuk:
Presynaptic autoregulation of norepinephrine release from sympathetic nerve fibres in the pineal gland of the domestic pig.
Pharmacological characterization of a2-adrenoceptors mediating this process
2002; 23 (suppl 1):111-117 pii:NEL230702A09 PMID:

Key words:
pig; pineal gland, sympathetic nervous system; neuronal uptake; norepinephrine release; a2-adrenoceptors; presynaptic regulation

Abstract
BACKGROUND: Norepinephrine is the main neurotransmitter controlling melatonin secretion in the mammalian pineal gland. Presynaptic autoregulation of norepinephrine release from the sympathetic nerve fibers in the pineal gland is poorly known.

METHODS: Uptake and depolarization-evoked release of 3Hnorepinephrine were investigated in vitro using pieces of the pig pineals. Specific antagonists and agonists of a2adrenoceptors were employed for the characterization of a2-adrenoceptors involved in the autoregulation of depolarization-evoked release of norepinephrine in the pig pineal.

RESULTS: The level of neuronal uptake of norepinephrine in the pig pineal was 3.5±0.9 pmol/h/mg of wet tissue and represented about 77% of the total tissue radioactivity. Potassium ions at concentration of 60 mM significantly evoked tritium release. This effect was abolished in the absence of extracellular Ca2+ and was diminished in the presence of Cd2+. Antagonists of a2-adrenoceptors increased depolarizationevoked tritium release. The order of its potency (based on pED30) was rauwolscine > phentolamine > BRL 44408 > WB 4101> RS 79948 = yohimbine >> prazosin >> imiloxan. a2-agonists decreased K+evoked release of tritium with the order of potency: UK14,304 > norepinephrine = guanfacine > oxymetazoline.

CONCLUSION: The processes of neuronal uptake and depolarization-evoked release of norepinephrine from the sympathetic nerve endings in the pig pineal gland have been demonstrated. The studies with the use of adrenergic antagonists and agonists indicate that the sympathetic nerve fibers in the pig pineal gland possess functional presynaptic a2adrenoceptors, which are involved in norepinephrine release inhibition. Due to the pharmacological properties these receptors closely resemble the subtype a2A.

Norepinephrine released from the sympathetic nerve fibers is considered as the main neurotransmitter regulating melatonin synthesis and therefore responsible for the nocturnal increase in the secretion of this pineal hormone.
Majority of our knowledge about the adrenergic regulation of the mammalian pinealocyte activity derives from the studies performed in the rat. Norepinephrine acts on the melatonin synthesis pathway in the rat pinealocytes via two postsynaptic adrenoceptors: a1 and b1 [1, 2]. The stimulation of b1-adrenergic receptors leads to the increase in cyclic AMP production, activation of transcription and translation of arylalkylamine Nacetyltransferase (enzyme limiting melatonin synthesis and secretion) and the inhibition of the proteosomal proteolysis of this enzyme [3, 4, 5]. The activation of a1adrenoceptors, which by itself does not alter the activity of adenylate cyclase and arylalkylamine N-acetyltransferase, potentiates b1-adrenergic stimulation of cAMP and melatonin production [1, 2]. The presence of postsynaptic a2-adrenoceptors functionally linked to membrane guanylate cyclase has been also demonstrated in the rat pineal gland [6, 7].
Investigations performed in different mammalian species provided evidence for species heterogeneity in the adrenergic regulation of melatonin secretion in mammals [8, 9, 10, 11, 12]. The interspecies differences concern both the adrenergic receptors involved in the regulation of the pinealocyte activity at postsynaptic level [8, 9] as well as the intracellular mechanisms, which control the rate of melatonin synthesis [10, 11, 12]. ... ...

The future of melatonin as a
therapeutic agent
pages: 118-121; pii:NEL230702X01; PMID:

Abstract
Report of the round table conference summarizing the International Symposium on "Melatonin: Clinical Significance and Therapeutic Applications" is presented in this article. Some sleep disorders and circadian rhythm disturbances are the widely accepted indications for melatonin treatment. However, other possibilities for use of melatonin in the therapy should be also taken into account, including a co-treatment in cancer patients and free radical-related diseases. All aspects of the possible therapeutic use of melatonin as well as its safety, dosage, side effects and contraindications are discussed herein based on the round table conference and they are presented in this paper.

Introduction
A round table discussion on the future of melatonin in the therapy was held at the end of the International Symposium in Polanica Zdroj (Poland) devoted to clinical significance and therapeutic application of melatonin. The authors were the active participants of this conference, and this article summarizes the round table discussion.
Melatonin, the main secretory product of the pineal gland was discovered by Lerner and coworkers [1] in 1958. Although the diversity of functions of melatonin is still under debate, numerous investigations performed since that time brought about significant progress in understanding the relevance of this substance in animals and in humans. Moreover, melatonin has become recently available in some countries (e.g. USA, Argentina, Poland, China) as either an OTC drug or food supplement.
There are some widely accepted indications for therapeutic use of melatonin but also perspectives for its broader use. Both the accepted indications and further perspectives are discussed in this article.

Generally accepted indications for therapeutic use of melatonin
Melatonin in sleep disorders ...
Melatonin in circadian clock disturbances ...

Other possibilities for therapeutic use of melatonin
Melatonin as free radical scavenger ....
Melatonin in aging and age-related diseases ...
Melatonin in clinical oncology ...
Melatonin in circulatory system diseases ...

NEUROENDOCRINOLOGY LETTERS (NEL)
including Psychoneuroimmunology, Neuropsychopharmacology,
Reproductive Medicine, Chronobiology and Human Ethology
ISSN 0172–780X

NEUROENDOCRINOLOGY LETTERS:
A peer-reviewed transdisciplinary Journal covering neuroendocrinology, psychoneuroimmunology and chronobiology. A peer-reviewed transdisciplinary Journal. RAPID publication of papers from basic and clinical research, review articles and state-of the-art.
E-mail: editor@nel.edu

NEUROENDOCRINOLOGY LETTERS (NEL)
including Psychoneuroimmunology, Neuropsychopharmacology,
Reproductive Medicine, Chronobiology and Human Ethology. ISSN 0172–780X

SUPPLEMENT 1, 2002
Ordering info HERE
(per article $35.00)

MELATONIN: Clinical Significance and
Therapeutic Application

ABSTRACTS
ORIGINAL ARTICLES
1 Pawlikowski et al | 2 Rimler et al | 3 Winczyk et al
4 Karasek et al | 5 Markowska et al | 6 Bortkiewicz et al
7 Karasek et al | 8 Ostrowska et al | 9 Lewczuk

10 ROUNDTABLE CONFERENCE REPORT:
by Karasek, Reiter, Cardinali, Pawlikowski

NEUROENDOCRINOLOGY LETTERS (NEL) including Psychoneuroimmunology, Neuropsychopharmacology, Reproductive Medicine, Chronobiology and Human Ethology. ISSN 0172–780X

SUPPLEMENT 1, 2002 Ordering info HERE (per article $35.00)

MELATONIN: Clinical Significance and Therapeutic Application

ABSTRACTS ORIGINAL ARTICLES 1 Pawlikowski et al | 2 Rimler et al | 3 Winczyk et al 4 Karasek et al | 5 Markowska et al | 6 Bortkiewicz et al 7 Karasek et al | 8 Ostrowska et al | 9 Lewczuk

10 ROUNDTABLE CONFERENCE REPORT: by Karasek, Reiter, Cardinali, Pawlikowski

NEUROENDOCRINOLOGY LETTERS (NEL)
including Psychoneuroimmunology, Neuropsychopharmacology,
Reproductive Medicine, Chronobiology and Human Ethology. ISSN 0172–780X

SUPPLEMENT 1, 2002
Ordering info HERE
(per article $35.00)

MELATONIN: Clinical Significance and
Therapeutic Application

ABSTRACTS
ORIGINAL ARTICLES
1 Pawlikowski et al | 2 Rimler et al | 3 Winczyk et al
4 Karasek et al | 5 Markowska et al | 6 Bortkiewicz et al
7 Karasek et al | 8 Ostrowska et al | 9 Lewczuk

10 ROUNDTABLE CONFERENCE REPORT:
by Karasek, Reiter, Cardinali, Pawlikowski