Melatonin can enhance and suppress constitutive protein secretion from murine melanoma M2R cells in vitro in a cholera-toxin (CTX) sensitive process. In a number of tissues melatonin has been shown to modulate cGMP levels. The involvement of cGMP in melatonin responses in the melanoma cells was investigated. The effects of melatonin on melanoma cells cGMP and cGMP-phosphodiesterase activity and the effects of cGMP analogs on the melatonin-mediated modulation of protein secretion were studied. Melatonin reduced cGMP levels in the melanoma cells. CTX treatment had a similar and non-additive effect. The effects of melatonin on protein secretion were abrogated by activation of cGMP-dependent protein kinases. In addition, melatonin inhibited cGMP phosphodiesterase activity in these cells. The data presented indicate that inhibition of cGMP via a CTX sensitive G protein may be a major signal transduction pathway used by melatonin in melanoma cells.

The hormone melatonin modulates constitutive protein secretion and inhibits cGMP in melanoma M2R cells via cholera-toxin (CTX) sensitive pathways. Activation by melatonin of CTX-substrates is due to enhancement of their ADP ribosylation. The possibility that ADP ribosylation was enhanced by elevation of NAD+ was studied. Melatonin enhanced NAD+ and decreased cADP-ribose in the cells, in a CTX independent pathway, indicating inhibition of nicotinamide adenine dinucleotide glycohydrolase (NADase). Dibutyryl cGMP (db-cGMP), which obviates the melatonin-induced decrease in cGMP and prevents the modulation of protein secretion, abrogated the enhancement of NAD+. cADP-ribose is involved in calcium homeostasis and its decrease may reduce intracellular Ca2+. The intracellular Ca2+ chelator BAPTA/AM mimicked and Ca2+ ionophores prevented the melatonin-induced inhibition of protein secretion. These data indicate for the first time hormonal modulation of NADase resulting in two signals: (1) enhancement of NAD+ which may explain the increase in ADP ribosylation and activation of CTX substrates leading to facilitation of protein secretion; (2) suppression of cell cADP-ribose and consequently intracellular Ca2+ which may explain the melatonin-induced inhibition of protein secretion.

The pineal hormone melatonin modulates constitutive protein secretion from murine melanoma M2R cells in vitro, in a cholera-toxin (CTX)-sensitive process, without effecting major changes in cAMP. The effects of melatonin on GTP binding proteins and putative CTX substrates in these cells were investigated. Melatonin enhanced GTPγ35S binding and the incorporation of 32P-P3-(4-azidoanilido)-P1-5′-guanosine triphosphate (Az-32P-GTP) into 94, 40 and 28 kilodalton proteins. Similar changes were induced by CTX treatment. In addition, melatonin enhanced ADP ribosylation of several proteins, among them 94 and 40 kilodalton bands, apparently at arginyl residues. CTX catalyzed the ADP ribosylation of 45 and 40 (both recognized by antibodies specific to the C-terminal peptide of the Gsα subunit) and 94 kilodalton proteins and attenuated melatonin's effect. The melatonin-mediated ADP ribosylation reactions were attenuated by nicotinamide which inhibits mono(ADP ribosyl)transferases and poly(ADP-ribose)synthetase, but not by 3-amino benzamide, a specific inhibitor of poly(ADP-ribose)synthetase. Nicotinamide but not 3-amino benzamide prevented the enhancement by melatonin of GTPγ35S binding. These results indicate that melatonin enhances protein ADP ribosylation and consequently GTP exchange in a number of CTX-sensitive G proteins. They demonstrate a novel route for concerted activation of multiple GTP binding proteins by a single hormone.

Melatonin has been found to inhibit or enhance the constitutive secretion of proteins from the cultured melanoma cells at nanomolar concentrations (0.5-10 nM), in a dose dependent manner. The amplitude and direction of the response were found to depend on cell density: melatonin inhibited the release early after plating or at low cell density, but facilitated the release later on, or at high cell density. To elucidate the involvement of G-proteins in these responses, the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP tau S; which was introduced into the cells during the process of permeabilization and resealing with ATP), aluminum fluoride, pertussis and cholera toxins on protein secretion from the cells were assessed in the absence and presence of melatonin. At low cell density, melatonin inhibited release, but paradoxically enhanced it when GTP hydrolysis was blocked (by GTP tau S or cholera toxin treatment). Aluminum fluoride and melatonin inhibited protein release in the absence or presence of GTP tau S. At high cell density, melatonin facilitated the release and so did GTP tau S, aluminum fluoride, their combination, and cholera toxin treatment. However, in the presence of the combination of GTP tau S, aluminium fluoride and melatonin, protein release was paradoxically inhibited. Similar treatment of the cells with pertussis toxin, did not affect the melatonin-mediated inhibition or facilitation. These results indicate that the effects of melatonin on protein secretion are mediated by at least one heterotrimeric G protein which belongs to the Gs class. In addition, melatonin can facilitate secretion via a cholera and pertussis toxins-insensitive mechanism which can be inhibited by aluminum fluoride. This effect is manifested when Gs is permanently activated (by GTP tau S or cholera toxin).

The pineal hormone melatonin modulates constitutive protein secretion from melanoma M2R cells. Nanomolar concentrations of melatonin inhibited protein secretion early after plating or at low cell density, but facilitated it late after plating or at high cell density. Inhibition by melatonin of adenylate cyclase is the best known downstream response to melatonin. We have therefore examined the involvement of cAMP in the melatonin-mediated modulation of protein secretion from the melanoma cells. Melatonin slightly but significantly reduced cell cAMP content when effecting inhibition and marginally increased cAMP levels when effecting facilitation of protein secretion. Dibutyryl cAMP abrogated the melatonin-mediated inhibition but not facilitation of protein secretion without affecting basal secretion. Accordingly, forskolin prevented the inhibitory action of melatonin on protein secretion without affecting basal secretion. The selective protein kinase A inhibitor H-89 did not alter the inhibitory effect of melatonin at low cell density and slightly facilitated secretion at high cell density with or without melatonin. Thus, melatonin's effects on protein secretion may not be mediated via cAMP. Nevertheless, changes in cAMP or protein kinase A activity can abrogate, or mask, the melatonin-mediated responses.