Cyclic AMP

Melatonin has inhibitory effects on cAMP in both systems. In the cultured hemip-ituitaries of neonatal rats, melatonin inhibits cAMP accumulation induced by GnRH or forskolin (30,31). Melatonin also decreases the basal concentration of cAMP in the cultured hemipituitaries. Dispersed pituitary cell cultures were later used to study inhibitory effects of melatonin and its derivatives on forskolin-induced cAMP accumulation (25; Figure 3). The effect of melatonin on cAMP is dose-dependent (EC50 = 0.15 nM) and mediated by PTX-sensitive G-protein.

In cultured SCN cells, melatonin inhibits the VIP-induced increase of cAMP (33; Figure 3). The melatonin effect is dose-dependent, EC50 is 0.21 nM. Melatonin has no effect on basal cAMP, however.

The molecular mechanism of the melatonin effect is not clear. Because melatonin inhibits cAMP accumulation in the pituitary even in the presence of phosphodies-terase inhibitor 3-isobutyl-1-methylxanthine it has been concluded that the indole inhibits adenylyl cyclase (25,30). However, no direct evidence showing this effect is available.

The melatonin-induced decrease of cAMP is not of primary importance for inhibition of LH-release. Melatonin inhibits the GnRH-induced LH release even in the presence of permeable cAMP derivative 8-bromo-CAMP. Melatonin induced decrease of intracellular free calcium ([Ca2+]i) is the most important signal for inhibition of LH release: when the decrease of [Ca2+]i is prevented by Ca2+ ionophore, melatonin does not inhibit LH release. Nevertheless, cAMP may be involved in transduction of the melatonin signal, because it stimulates Ca2+ influx in subpopulations of gonadotrophs. Melatonin-induced decrease of cAMP may thus inhibit Ca2+ influx

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Melatonin (M)

Figure 3. Dose-dependent effect of melatonin on cAMP accumulation. In the pituitary cells (left part) forskolin (10 ^M) was used to increase cAMP accumulation in the presence of various concentration of melatonin and cAMP accumulation in the cells was determined after 30 min incubation. Suprachiasmatic cells (right part) were incubated in the presence of vasoactive intestinal peptide (VIP, 100 nM) together with various concentration of melatonin for 90 min and cAMP concentration in the incubation medium was measured. Each point represents the mean (±SEM) from 4 to 6 independent cultures.

resulting in inhibition of LH release (26). The involvement of cAMP in the inhibitory effect of melatonin on AVP release from SCN neurones is currently being studied. Our preliminary data indicate that an increase of cAMP induced by forskolin stimulates AVP release from the neurones. Melatonin may thus inhibit AVP release from SCN neurones via the decrease of cAMP.

3.3. Intracellular Free Ca2+

Because the GnRH-induced increase of [Ca2+]i is the primary signal for release of LH from gonadotrophs (3,4,21), melatonin effects on [Ca2+]i have been studied. GnRH induces [Ca2+]i increase by two mechanisms. Initially Ca2+ is released from intracellular IP3-sensitive stores which is followed by Ca2+ influx through voltage-sensitive channels (7,21). Our data indicate that melatonin may inhibit both pathways.

When gonadotrophs are incubated in Ca2+-free medium, the GnRH-induced [Ca2+]i increase is mediated by mobilisation from intracellular stores. Because in the neonatal rat gonadotrophs the Ca2+ stores are quite limited, the Ca2+ spike is transient and followed by rapid decrease to basal levels in most of the cells. In the presence of melatonin, the GnRH-induced Ca2+ spike is inhibited in 30% of gonadotrophs (19). This finding indicates, that melatonin inhibits GnRH-induced Ca2+ mobilisation from intracellular stores in about 1/3 of the gonadotrophs.

How melatonin inhibits the Ca2+ mobilisation is not clear. Three possible mechanisms could be involved: 1) inhibition of phospholipase C and IP3 formation; 2) inhibition of IP3 binding on its receptor in endoplasmic reticulum or inhibition of the channel opening; 3) increased Ca2+ clearance from cytosol. Based on indirect evidence, the most likely mechanism is inhibition of phospholipase C. This enzyme metabolises phos-phatidylinositol bisphosphate into diacylglycerol and inositol trisphosphate (IP3), which in turn induces the release of Ca2+ from endoplasmic reticulum (1). Melatonin has been shown to inhibit the GnRH-induced formation of diacylglycerol in the gonadotrophs (32) and may also inhibit the IP3 accumulation (J.Vanecek, unpublished data).

Apart from inhibiting Ca2+ mobilisation, melatonin also blocks Ca2+ influx through voltage-sensitive channels (27,28). In the neonatal rat gonadotrophs cultured in Ca2+-supplemented medium, the GnRH administration induces Ca2+ spike followed either by a sustained plateau or by calcium oscillations (19). Melatonin added after the GnRH-induced spike decreases [Ca2+]i in about 50% of the gonadotrophs. The mela-tonin effect has been mimicked by inhibitor of voltage-sensitive Ca2+ channels vera-pamil and in Ca2+-free medium the melatonin effect is abolished. These observations indicate that melatonin inhibits Ca2+ influx through voltage-sensitive channels. Mechanism of the effect may involve hyperpolarization of plasma membrane, because mela-tonin has been shown to increase membrane potential in neonatal rat gonadotrophs (28,29). Hyperpolarization closes the voltage-sensitive channels and inhibits the Ca2+ influx.This pathway may represent the most important mechanism for inhibition of LH release by melatonin. In the presence of the L-channel agonist Bay K, the melatonin inhibitory effect on LH release is markedly reduced while the inhibitor of voltage-sensitive Ca2+ channels nifedipine inhibits GnRH-induced LH release to a similar degree as melatonin. Moreover, the effects of melatonin and nifedipine are not additive. This conclusion correlates with the known important role of intracellular Ca2+ in the regulation of LH release.

Melatonin may affect [Ca2+]i also in the SCN neurones. Our preliminary data indicate that VIP induces [Ca2+]i increase in about 14% of the SCN cells. In some of them, the [Ca2+]i increase is blocked in the presence of melatonin. However, these studies are complicated, because the VIP response desensitises rapidly and only a few of the cells are sensitive to melatonin. Nevertheless, taken all together our observations indicate that in gonadotrophs and in SCN neurones melatonin has similar effects and may act through similar mechanisms.

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