Results And Discussion

The nocturnal maxima of melatonin-deacetylating aryl acylamidase activity, reflecting 5MT formation, and of 5MT concentration are found in identical phase positions; moreover, the circadian glow peak appears at the time of highest 5MT levels (Figure 1). Therefore, a substantial precondition is fulfilled for an involvement of 5MT in the generation of the glow peak. Inhibition of indoleamine biosynthesis by blocking tryptophan hydroxylase leads to a marked suppression of the glow peak, as demonstrated by three different inhibitors, p-chlorophenylalanine, p-fluorophenylalanine and 5-fluorotryptophan (Figure 1). We had previously shown that a glow maximum suppressed by p-chlorophenylalanine can be restored by 5-hydroxytryptophan (8). Here we demonstrate that such a restoration is also possible with MLT and 5MT (Figure 1). In the case of 5MT, near-physiological concentrations are sufficient for this effect. Higher doses of MLT are required, in order to generate stimulatory amounts of 5MT, which is readily catabolized to 5-methoxytryptophol (1). More extended studies using all three inhibitors and all intermediates (not shown) revealed that any indolic metabolite which can be converted to 5MT is capable of restoring the glow peak; however, other products such as substituted tryptophols, not leading to 5MT formation, are ineffective.

We have recently shown that both MLT and 5MT strongly decline in Gonyaulax exposed to oxidative stress, as induced by differently acting substances, such as (a) hydrogen peroxide, a source of hydroxyl radicals, (b) paraquat (PQ), a herbicide generating superoxide anions, and (c) buthionine sulfoximine (BSO), an agent decreasing reduced glutathione (9). While hydrogen peroxide is highly toxic to Gonyaulax (7), the weaker stressors PQ and BSO are tolerated by the dinoflagellate up to remarkably high concentrations. Here we demonstrate that both PQ and BSO also suppress the nocturnal glow peak (Figure 1). Again, the maximum can be restored by MLT, although this is not a stimulator of bioluminescence per se. The restoration is more clearly expressed in the case of PQ, as compared to BSO treatment. Also 5MT is capable of enhancing bioluminescence in PQ- or BSO-treated cells (Figure 1); however, concentrations higher than physiological are required (30 or 10 (M, respectively), presumably because of the continuous presence of the stressors leading to permanent intracellular oxidative destruction of the indoleamine. For this reason, increases of bioluminescence in a phase position similar to that of the normal glow peak are only seen when 5MT is given shortly before this phase. On the other hand, its precursor MLT, which does not stimulate bioluminescence directly and, therefore, can be given at higher concentrations (250 or 100(M, respectively), is also effective when added much earlier, at the

Figure 1. 5-Methoxyindoles and the expression of the glow peak. Left column: Coincidence of maxima of aryl acylamidase (AAA), 5MT and spontaneous bioluminescence (BL); experiments in LD. 2nd to 4th columns: Suppression of the glow peak by inhibitors of tryptophan hydroxylase, by paraquat and by buthionine sulfoximine, and its restoration by MLT and 5MT: experiments in DD. RBLU—relative bioluminescence units. Black bars: dark time. Vertical lines: s.e.m. Other abbreviations: C — control: CPA—p-chlorophenylalanine 0.1 mM: FPA—p-fluorophenylalanine 0.1 mM: FTP—5-Huorotryptophan 1 mM; PQ1—paraquat 1 mM; PQ2—paraquat 2mM; BSO—buthionine sulfoximine 10 mM: doses of MLT—2nd and 4th columns: 0.1mM, 3rd column: 0.25 mM; doses of 5MT—2nd column: 1|iM. 3rd column: 30 |iM, 4th column: 10 |iM.

Figure 1. 5-Methoxyindoles and the expression of the glow peak. Left column: Coincidence of maxima of aryl acylamidase (AAA), 5MT and spontaneous bioluminescence (BL); experiments in LD. 2nd to 4th columns: Suppression of the glow peak by inhibitors of tryptophan hydroxylase, by paraquat and by buthionine sulfoximine, and its restoration by MLT and 5MT: experiments in DD. RBLU—relative bioluminescence units. Black bars: dark time. Vertical lines: s.e.m. Other abbreviations: C — control: CPA—p-chlorophenylalanine 0.1 mM: FPA—p-fluorophenylalanine 0.1 mM: FTP—5-Huorotryptophan 1 mM; PQ1—paraquat 1 mM; PQ2—paraquat 2mM; BSO—buthionine sulfoximine 10 mM: doses of MLT—2nd and 4th columns: 0.1mM, 3rd column: 0.25 mM; doses of 5MT—2nd column: 1|iM. 3rd column: 30 |iM, 4th column: 10 |iM.

time of transfer to DD (Figure 1). In this case, MLT will be preferentially converted to 5MT not before the second half of subjective night, due to the phasing of aryl acy-lamidase, and therefore lead to a glow peak in approximately correct phase position.

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