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Melatonin was found to have a small inhibitory effect on tyrosinase activity and a slight stimulatory action on dopachrome tautomerase activity in B16 mouse melanoma cells. These effects were time and dose dependent, with the maximal response being observed after 24-48 h treatment and at concentrations of melatonin higher than the physiologic levels of the circulating hormone. Although these effects on the melanogenic activities were modest, incubation of melanocytes with melatonin prior to the addition of the melanotropin mediated a dramatic inhibition of a-melanocyte-stimulating-hormone-(a-MSH)-induced melanogenesis. This inhibitory effect was evident at melatonin concentrations as low as 10 nM. Inhibition was nearly total at 0.1 mM melatonin, even at high concentrations of a-MSH (1 pM).The inhibitory effect of melatonin on a-MSH stimulation of melanogenesis was investigated. Melatonin appeared to act at least at two stages. Pharmacological concentrations of melatonin diminished the number of a-MSH receptors to about 75% of the control values without an apparent effect on receptor affinity, as determined by receptor-binding studies using '251-[N-Leu4-~-Phe7]a-MSH as a probe. Physiological concentrations of melatonin also appeared to interfere with the intracellular events coupling increased CAMP levels and induction of the c locus tyrosinase, since it strongly inhibited the theophyllinemediated stimulation of melanogenesis. The inhibiton of tyrosinase stimulation was higher in the microsoma1 than in the melanosomal fractions of cells which were treated with melatonin, then exposed to either a-MSH (1 pM) or theophylline (1 mM), suggesting that one of the main effects of melatonin might be inhibition of the induction of tyrosinase de novo synthesis.Keywords : melatonin ; a-melanocyte-stimulating hormone ; tyrosinase ; tyrosinase-related proteins ; melanogenesis.Mammalian pigmentation is the result of melanin production by the epidermal melanocytes. Within melanocytes, melanin synthesis proceeds in specialized organelles, the melanosomes, through a series of coupled enzymic and chemical reactions (reviewed in Prota, 1992). The two initial steps of the pathway, rate-limiting hydroxylation of tyrosine to yield ~-3P-dihydroxyphenylalanine (L-Dopa) and L-Dopa oxidation to L-dopaquinone, are catalyzed by tyrosinase. These two reactions can also be catalyzed, although with lower efficiency, by the tyrosinaserelated protein-1 (TRP1; Jimenez et al., 1991 ; JimCnez-Cervantes et al., 1994) a protein encoded by the b Locus (Jackson, 1988). At least one other enzymic protein, dopachrome tautomerase (Aroca et al., 1990), has been shown to be involved in regulation of the pathway. This enzyme catalyzes the tautomerization of L-dopachrome, the product of spontaneous evolution

Seven experiments were performed to investigate the sensitivity of the hamster pineal gland to exogenously administered norepinephrine (NE). In these studies NE (1 mg/kg) administration was preceded (10 min earlier) by the injection of the catecholamine uptake inhibitor desmethylimipramine (DM I; 5 mg/kg). When DMIand NE were given at night, the hamsters were exposed to light to depress pineal N-acetyltransferase activity and melatonin values to low levels; the drugs were then given 20 (DMI) and 30 (NE) min later, and the subsequent changes in pineal N-acetyltransferase and melatonin were monitored. The combination of DMI and NE administration anytime during the normal light period or during the first 4 h of the normal dark period failed to stimulate either pineal N-acetyltransferase activity or melatonin levels. Conversely, DMI followed by NE (injected either intraperitoneally or subcutaneously) in the second half of the dark phase typically stimulated pineal melatonin production. Likewise, the NE agonist isoproterenol promoted pineal melatonin production only in the latter half of the dark phase. If hamsters were exposed to continual light at night or if they were superior cervical ganglionectomized, a procedure which sympathetically denervates the pineal gland, the stimulatory effect of NE on melatonin production was significantly suppressed. Thus, the hamster pineal gland is sensitive to NE only during the latter half of the normal dark period and both darkness and an intact sympathetic innervation to the pineal gland are required for the gland to develop maximal sensitivity to the catecholamine. Also, the hamster pineal seems not to exhibit a supersensitivity response to NE following a period of reduced exposure to the catecholamine. The normal nocturnal rise in melatonin production in the hamster pineal gland seems to be determined by two parameters: an increased production and secretion of NE by sympathetic nerve endings in the pineal at night and (2) an increased sensitivity of the β-receptors on the pinealocyte membranes to NE during the late dark phase.

Recently, it was shown that a 1.5-ml subcutaneous saline injection depressed N-acetyltransferase (NAT) activity and melatonin content in the rat pineal gland at night. The present studies were undertaken to determine if another perturbation, swimming, could duplicate this response. Rats swam at 23.10 h (lights out at 20.00 h) for 10 min and were killed 15 and 30 min after the unset of swimming. Pineal NAT activity was found to be unaffected while melatonin content was depressed dramatically. Hydroxyindole-O-methyltransferase (HIOMT) activity as well as the content of serotonin (5HT), 5-hydroxytryptophan (5HTP) and 5-hydroxyindoleacetic acid (5HIAA) were not changed by this treatment. In a second study, pineal melatonin again was depressed without a concomitant drop in NAT activity. Mean serum melatonin at 15 min after onset of swimming was increased although the rise was not statistically significant. In the final study, it was found that NAT activity was slightly increased in intact rats and unchanged in adrenalectomized rats at 7 min after swimming onset. At 15 min both intact and adrenalectomized animals had NAT activity values similar to those of controls. Pineal melatonin content in intact and adrenalectomized rats plummeted to 50% of control values at 7 min and fell further to 25% at 15 min. While the rate of melatonin synthesis was not directly measured, lack of change in the activities of the enzymes involved in melatonin synthesis and the contents of two melatonin precursors suggests that swimming depresses pineal melatonin content by enhancing melatonin efflux from the gland.

The cytosolic enzymes asymmetrical diadenosine tetraphosphate hydrolase (EC 3.6.1.17, Ap R Aase) and diadenosine triphosphate hydrolase (EC 3.6.1.29, Ap Q Aase) are inhibited competitively by suramin. Ap R Aase and Ap Q Aase were assayed in cytosolic rat brain extracts using fluorogenic analogues of the respective substrates diadenosine tetraphosphate (Ap R A) and diadenosine triphosphate (Ap Q A). K i values for suramin as inhibitor of Ap R Aase and Ap Q Aase were 5U10 3T M and 3U10 3U M, respectively. Results indicate that suramin or suramin-like derivatives may be useful tools to investigate diadenosine polyphosphate cleaving enzymes and that the intracellular diadenosine polyphosphate metabolism may be a pharmacological target of suramin with biological and clinical implications.z 1998 Federation of European Biochemical Societies.