M. G. M. Balemans

Circadian rhythmicity of the activity of hydroxyindole-o-methyl transferase (HIOMT) in the formation of melatonin and 5-methoxytryptophol in the pineal, retina, and harderian gland of the golden hamster

The day and night rhythms in the activity of HIOMT in the formation of melatonin and of 5-methoxytryptophol have been determined in the pineal, retina and Harderian gland of the adult male golden hamster. In all hamsters used there was no detectable HIOMT activity in the deep pineal. In the superficial pineal HIOMT activity, involved in the synthesis of melatonin (Mel), was observed to be high at the end of the dark period and at the middle of the light period. Considering the HIOMT activity involved in the production of 5-methoxytryptophol (5-MTL), an increase in 5-MTL synthesis was observed only during the light period. Comparing the peak of Mel-production with that of 5-MTL it appears that during the light period the pineal produces more 5-MTL than Mel. In the Harderian glands, the circadian course of HIOMT activity involved in the synthesis of Mel seems to run parallel to that of the enzyme implicated in 5-MTL synthesis, both being stimulated at the end of the dark period. The activity of HIOMT in 5-MTL production is, however, always approximately 2 times higher than for Mel synthesis. In the retina the synthesis of Mel and 5-MTL is not significantly higher during the dark period than during the light period. However, the production of 5-MTL is larger than that of Mel. It appears that (1) with the exception of the end of the dark period, the extra-pineal synthesis of Mel and 5-MTL is always higher than that in the pineal; (2) the circadian synthesis of 5-methoxyindoles is different in each organ, and (3) in the pineal the circadian activity of HIOMT involved in 5-MTL formation is different from that of the same enzyme involved in the formation of Mel. The results are discussed.

Preliminary investigations on melatonin and 5-methoxytryptophol synthesis in the pineal, retina, and Harderian gland of the mole rat and in the pineal of the mouse “eyeless”

Hydroxyindole-O-methyltransferase (HIOMT) activity for the synthesis of melatonin and 5-methoxytryptophol, both 5-methoxyindoles, was measured in the pineal, the Harderian gland and the retina of the mole rat and in the pineal of the mouse “eyeless”. In the pineal and the Harderian gland of the mole rat a larger amount of 5-methoxytryptophol than of melatonin is synthesized. 5-Methoxyindole synthesis is extremely high in the Harderian gland, whereas in the retina HIOMT activity is low and variable. In the pineal of the mouse “eyeless”, a low 5-methoxyindole synthesis showing no circadian rhythm is demonstrated. It is concluded that, besides the generally accepted regulation of the indole metabolism by light, in species with atrophied eyes having Harderian glands (mole rat) and in species without eyes other factors than light might be responsible for the indole metabolism in the pineal gland.

Day/night rhythmicity in the methylating capacities for different 5-hydroxyindoles in the pineal, the retina and the Harderian gland of the golden hamster (Mesocricetus auratus) during the annual seasons.

In the pineal, the Harderian gland and the retina of the golden hamster the day/night capacity for the synthesis of different methoxyindoles is investigated under natural conditions for one 24-hour period in the months of December, March and June. The amounts of the methoxyindoles and the diurnal rhythms in the synthesis are different in the various months during which the tests were performed. There is a striking increase in the synthesis of melatonin and methoxytryptophol in all three organs in June as compared with December. Equally striking is the high synthesis of methoxytryptamine in the pineal in June, whereas this compound was not formed in the Harderian gland and the retina in this month. Methoxytryptophan synthesis was not observed in June in any of the three organs. Methoxyindoleacetic acid rhythmicity shows a pattern more or less identical to that of melatonin and methoxytryptophol. A high synthesis exists at the end of the June day, but greater fluctuations during the days of the two other months. Acetylmethoxytryptophol is synthesized in the pineal during the night in March, but in the late afternoon in June. The largest quantity of this compound is found in the Harderian gland in December, in the pineal in March and in the retina in June. The largest amounts of melatonin and methoxytryptophol are synthesized when gonadal weight is largest. The possible correlation between the amounts synthesized, the rhythmicities in synthesis and the reproductive system is discussed.

Estimation of the methylating capacity of the pineal gland. With special reference to indole metabolism.

In order to obtain more information on the methylating capacity of the pineal gland, a method determining the formation of different 5-methoxyindoles in the pineal gland was developed. The method depends on measuring the incorporation of labelled methyl groups into the various hydroxyindoles present in the pineal gland, after incorporation of pineal tissue with labelled S-adenosyl methionine. Hydroxyindoles were not added to the incubation medium. After incubation thin-layer chromatography was performed with pineal tissue together with the incubation medium; the spots were scraped and counted.

The pineal gland of the mole (Talpa europaea L.) VII. Activity of hydroxyindole-O-methyltransferase (HIOMT) in the formation of 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3-acetic acid, 5-methoxytryptophol and melatonin in the eyes and the pineal gland

Using a method in which no substrate is added to the incubation medium, the capacity of HIOMT to synthesize 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3-acetic acid, 5-methoxytryptophol and melatonin has been determined in the pineal and the eyes of the mole, a mammal having an atrophied visual system. The results demonstrate that the indoleamine metabolism in the retina is similar to the indoleamine metabolism in the pineal. Moreover, in all animals studied both eyes together synthesize 2 to 30 times more of 5-methoxyindoles than the pineal, a result which proves again that the pineal is not the only and not always the most important source of 5-methoxyindoles. With the exception of 5-methoxyindole-3-acetic acid, the synthesis of which is practically neglectable, the production of all other 5-methoxyindoles in the pineal as well as in the retinae is always larger than that of melatonin. In the pineal, 5-methoxytryptophan, for example, is synthesized in a quantity which is 60 to 170 times larger than that of melatonin, while in the retinae the synthesized amount of 5-methoxytryptophan is even 60 to 1000 times larger than that of melatonin.

Seasonal variations in HIOMT activity during the night in the pineal gland of 21 day old male Wistar rats.

In the pineal of 21-day old male Wistar rats hydroxyindole-O-methyl-transferase (HIOMT) activities involved in the synthesis of several 5-methoxyindoles were determined during the night in April, June, October and January. A high HIOMT activity for the synthesis of melatonin/5-methoxytryptophol was determined in the months of January and April. In June and October a decrease was observed. The activity maxima coincide with peaks of activity found for the synthesis of 5-methoxytryptophan. Synthesis of 5-methoxytryptamine occurred only in June and October, whereas the synthesis of 5-methoxyindole-3-acetic acid occurred only in January. From these results it may be concluded that January and April are the most active months of those tested for the melatonin/5-methoxytryptophol synthesis in the rat pineal gland. A possible physiological role of the 5-methoxyindoles other than melatonin and 5-methoxytryptophol is discussed.

Estimation of the methylating capacity in the pineal gland of the rat with special reference to the methylation of N-acetylserotonin and 5-hydroxytryptophol separately.

In the present paper, an extension is presented of an earlier described method, by which the methylating capacity of the pineal gland can be determined. Supplementary to the earlier method, the synthesis of melatonin and 5-methoxytryptophol can now be qualified and quantified separately.

Effect of injecting 5-methoxy indoles, pineal compounds, on testicular weight of white leghorn cockerels (Gallus domesticus L.)

White leghorn cockerels of the same age with combs of about equal size and raised under identical conditions were injected with melatonin, 5-methoxytryptophol, or with 5-methoxytryptophol in combination with melatonin. Melatonin, injected in animals with small comb sizes, has no effect on testicular growth, whereas an inhibiting effect is noticed in animals with larger comb sizes. The 5-methoxytryptophol treated animals demonstrated stimulated testicular growth, which can be interpreted as an acceleration of the rhythm of growth. The stimulatory effect on testicular growth is more distinct if 5-methoxytryptophol is injected in combination with melatonin.

Day and night rhythms in the methylation of N-acetylserotonin/5-hydroxytryptophol in the pineal gland of male rats of different ages

Next to a night rhythm of the methylation of N-acetylserotonin/5-hydroxytryptophol the presence of a daytime rhythm could also be established. Rhythmicity was studied in May during the night and in June during daytime in 21, 42 and 70 days old male Wistar rats. In 21 days old rats, moderate HIOMT activity was observed from 12 p.m.–4 a.m. In rats aged 42 and 70 days HIOMT activity was increased showing a peak at 4 a.m. In September this night maximum is observed at 12 p.m. in rats, aged 42 days. This points to the presence of a seasonal change in HIOMT rhythmicity during the night. During daytime a moderate HIOMT activity is present, which reaches a maximum at 2 p.m. in the 21 and 42 day old animals, while in the adult 70 days old rats activity starts to increase at 2 p.m. probably reaching a maximum at 6 p.m.

Methoxyindole synthesis in the retina of the frog (rana esculenta) during a diurnal period

In the present study, the synthesis of methoxyindoles in the neural part and in the pigment epithelial layer of the retina of the frog eye was investigated on the basis of naturally occurring substrate at regular intervals during a 24 hour period. Melatonin, 5-methoxytryptophol and 5-methoxyindole acetic acid were synthesized by the neural part of the retina only, while 5-methoxytryptamine and 5-methoxytryptophan were produced by both, the neural part of the retina and the pigment epithelium. The synthesis of melatonin and of 5-methoxytryptamine showed a diurnal rhythmicity. The results obtained clearly indicate that another cell type,i.e. pigment cells, is involved in indole metabolism besides photoreceptor elements. A possible functional relationship between different methoxyindoles and different retino-motor processes in the amphibian eye is discussed.