Melatonin Effects

The pineal hormone melatonin is involved in photoperiodic regulations of reproductive functions and in entrainment of daily rhythms. In order to determine general intracellular mechanisms of melatonin action, we have compared melatonin effects in two different cell types. Melatonin acts through the specific high-affinity membrane receptors (K ~ 10-11 M) (22,24,36,38). Distribution of the melatonin receptors has been determined by in vitro autoradiography using 125I-melatonin. Melatonin receptors are present in discrete areas of the rat brain. High density of the receptors has been found in suprachiasmatic nuclei of the hypothalamus, in area postrema and in pars tuberalis of the pituitary (23,37). The receptor density in the rat pars distalis is age-dependent: it is about 30 fmol per mg of protein on embryonic day 20 and postnatal day 1, but within 30 postnatal days decreases 10 times, i.e. below 3 fmol/mg protein (23).

We have studied melatonin effects and their intracellular mechanisms in neonatal rat anterior pituitary and suprachiasmatic nuclei (SCN). In both cases, the primary cell cultures were used in our experiments. There are several similar aspects of mela-tonin action in both cell types. Melatonin has generally inhibitory effects: it inhibits release of luteinizing hormone (LH) from the pituitary gonadotrophs and release of vasopressin (AVP) from the SCN neurones (12,27,35). Moreover, melatonin inhibits cAMP accumulation in both systems. On the other hand, there are also specific effects of melatonin in each of the systems.

2.1. Anterior Pituitary

The release of luteinizing hormone from gonadotrophs is low under basal conditions and is markedly stimulated by GnRH. Melatonin has no effect on basal LH release, but it inhibits secretion of LH induced by GnRH (9; Figure 1). The melatonin effect on LH release was first described with cultured neonatal rat pituitaries and later confirmed using dispersed cells in culture (11,27). Inhibitory effect of melatonin on GnRH-induced LH release has been also observed in vivo (10).

Melatonin inhibits the effects of GnRH in a dose-dependent manner. A minimal inhibition of LH-release is seen with 10-10 and the maximal effect is attained with 10-8 to 10-7 M melatonin (9). The melatonin effect is specific, the order of potency of various indoles is 2-iodomelatonin > melatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-methoxytryptamine >> 5-hydroxytryptamine.

Melatonin inhibits not only release of LH but also of FSH from gonadotrophs (14). Melatonin acts probably directly on gonadotrophs, because it inhibits GnRH-induced LH release from enriched gonadotroph fraction (11). A melatonin effect on other cell types in the pituitary may not be completely excluded although it has not been possible to demonstrate any effect of melatonin on release of other pituitary hormones (14).

The inhibitory effect of melatonin on LH- and FSH-release is age dependent (9,13). While in 4 to 8-day-old rats, melatonin inhibits GnRH-induced LH release by about 60%, the melatonin effect gradually decreases starting day 10 and disappears almost completely after day 15 of age. The developmental changes of melatonin

Gnrh And Melatonin

Figure 1. Melatonin effect on GnRH-induced LH release from cultured pituitary cells of neonatal rat. The primary cell cultures were incubated for 3 hr in the presence of various concentration of melatonin and 2 nM GnRH. LH release into the incubation medium was determined by radioimmunoassay. Each point represents the mean (±SEM) from at least 3 independent cultures.

Figure 1. Melatonin effect on GnRH-induced LH release from cultured pituitary cells of neonatal rat. The primary cell cultures were incubated for 3 hr in the presence of various concentration of melatonin and 2 nM GnRH. LH release into the incubation medium was determined by radioimmunoassay. Each point represents the mean (±SEM) from at least 3 independent cultures.

potency correlate with the postnatal changes of the melatonin receptor density in the rat pituitary (23).

2.2. Suprachiasmatic Nuclei

Cultured hypothalamic slices containing SCN have been shown to release vasopressin in a circadian fashion (5). Later it has been found that dispersed suprachiasmatic neurones also show a circadian rhythm in AVP release (16,39). The release is low during subjective night and increases during subjective day peaking around circa-dian time (CT) (6). Melatonin has inhibitory effects on the spontaneous AVP release (35; Figure 2). Melatonin administered early in the morning delays the spontaneous increase of AVP release and decreases its amplitude. Melatonin added at the time of the peak, advances and accelerates the decrease of AVP release.

The inhibitory effect of melatonin is time dependent. When applied during subjective day, melatonin decreases the spontaneous AVP release by about 50%. However, markedly smaller inhibition is seen after melatonin administration at night and melatonin has no effect when applied around midnight.

Vasopressin release may be increased by vasoactive intestinal peptide (VIP). Melatonin also inhibits the VIP-induced AVP release induced from cultured SCN neurones (35). The melatonin effect is dose-dependent, inhibition starts at 10-10M and maximal effect is reached at 10-8 to 10-7 M melatonin. EC50 is about 0.4 nM.

The effects on AVP release correlate with melatonin effects on spontaneous electric activity of the SCN neurones. The electric activity of SCN neurones in cultured hypothalamic slices shows a circadian rhythm peaking at noon and decreasing during

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Figure 2. Melatonin effect on spontaneous release of vasopressin from cultured suprachiasmatic neurones. Two primary cultures of the neurones were incubated for 60 hr. The incubation medium was changed every 3 hr and vasopressin concentration determined by radioimmunoassay. Melatonin (100 nM) was added during the period indicated by the horizontal line to the culture No. 2(Y). • control, untreated culture.

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Figure 2. Melatonin effect on spontaneous release of vasopressin from cultured suprachiasmatic neurones. Two primary cultures of the neurones were incubated for 60 hr. The incubation medium was changed every 3 hr and vasopressin concentration determined by radioimmunoassay. Melatonin (100 nM) was added during the period indicated by the horizontal line to the culture No. 2(Y). • control, untreated culture.

late subjective day and night (6). Addition of melatonin decreases the frequency of the spontaneous firing (18,20). The effect is rapid, reversible and time-dependent. The maximal inhibitory effect is seen between CT 9 and CT 15, when most of the cells are inhibited by melatonin. At other times, the majority of the cells is unresponsive to melatonin.

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