Regulation of melatonin synthesis in rainbow trout (Oncorhyncus mykiss) pineal organs: effects of calcium depletion and calcium channel drugs.

The effects of calcium depletion and of three calcium channel drugs on melatonin synthesis in pineal organs of rainbow trout (Oncorhyncus mykiss) were examined. Dark-induced melatonin synthesis was inhibited by calcium depletion, by treatment with nitrendipine (NTR), an antagonist of the L-type voltage-sensitive calcium channel, and by treatment with omega-Conotoxin GVIA, an antagonist of the N-type voltage-sensitive calcium channel. Bay K 8644, an agonist of the L-type channel, had no significant effect on pineal melatonin synthesis. These data represent evidence that calcium entry into trout pineal photoreceptor cells through voltage-sensitive calcium channels is important in the maintenance of dark-induced melatonin synthesis and that light's inhibitory effect on melatonin synthesis may be mediated by closure of these channels. NTR-induced inhibition of dark-induced melatonin synthesis was abolished when dibutyryl cyclic AMP (db-cAMP) was administered to NTR-treated pineal organs, suggesting that calcium acts upstream of cAMP in regulating melatonin synthesis.

Regulation of melatonin production by pineal photoreceptor cells: role of cyclic nucleotides in the trout (Oncorhynchus mykiss).

The light/dark cycle influences the rhythmic production of melatonin by the trout pineal organ through a modulation of the serotonin N-acetyltransferase (NAT) activity. In static organ culture, cyclic AMP (cAMP) levels (in darkness) and NAT activity (in darkness or light) were stimulated in the presence of forskolin, isobutylmethylxanthine, or theophylline. Analogues of cAMP, but not of cyclic GMP, induced an increase in NAT activity. Light, applied after dark adaptation, inhibited NAT activity. This inhibitory effect was partially prevented in the presence of drugs stimulating cAMP accumulation. In addition, cAMP accumulation and NAT activity increase, induced by forskolin, were temperature dependent. Finally, melatonin release, determined in superfused organs under normal conditions of illumination, was stimulated during the light period of a light/dark cycle by adding an analogue of cAMP or a phosphodiesterase inhibitor. However, no further increase in melatonin release was observed during the dark phase of this cycle in the presence of the drugs. This report shows for the first time that cAMP is a candidate as intracellular second messenger participating in the control of NAT activity and melatonin production by light and temperature.

Examination of in vitro melatonin secretion from superfused trout (Salmo gairdneri) pineal organs maintained under diel illumination or continuous darkness.

Melatonin secretion was measured from rainbow trout (Salmo gairdneri) pineal organs maintained individually under flow-through whole organ culture (superfusion) conditions. Radioimmunoassay of perfusate fractions collected during controlled photic conditions demonstrated that melatonin secretion in vitro remained basal during the photophase and underwent increases in titer during the scotophase. While amounts of melatonin (mel) secreted were characteristic of individual pineal organs, photophase values ranged between 0.25 and 0.75 ng mel/ml and scotophase values ranged from 6 to 10 ng mel/ml of perfusate. Diel melatonin secretion profiles reflected the illumination regimen, with light associated with low melatonin titer in the perfusate and darkness associated with high titer. Light pulses during a normal scotophase resulted in a depression in melatonin secretion regardless of whether it was administered early or late in the dark period. Pulses of darkness given early or late in a normal photophase resulted in increased melatonin secretion. Superfused trout pineal organs did not display endogenous rhythmicity in melatonin secretion when subjected to prolonged exposure to continuous darkness (DD), whether first exposed to entraining light/dark (LD) cycles prior to DD or exposed to DD at the initiation of superfusion. In both studies, elevated melatonin secretion gradually declined over time. But exposure to a 4:4LD cycle after DD resulted in decreased (with light) and increased (with darkness) melatonin secretion. These results demonstrate that the trout pineal organ can be maintained for extended periods of time in superfusion culture, that the trout pineal organ is very responsive to light or dark for regulating melatonin synthesis, and that an endogenous rhythm in melatonin synthesis when organs were maintained in DD was not detectable.

Characteristics and non-rhythmicity of retinal hydroxyindole-O-methyltransferase activity in trout (Salmo gairdneri).

The validation and optimization of an hydroxyindole-O-methyltransferase (HIOMT) activity assay system for trout ocular enzyme was undertaken. High-performance liquid chromatographic analysis, cross-validated with a standard thin-layer chromatographic technique verified that HIOMT activity was present in trout ocular tissue by identifying the 3H-melatonin end product. This activity was observed in the neural retina and was restricted to those fractions containing the cytosolic component. The enzyme displayed bimodal pH optima, one at pH 7.6, the other at 8.4. N-Acetylserotonin was O-methylated preferentially and a Km of 4.5 X 10(-7) M was calculated for this substrate. The S-[3H]adenosylmethionine Km was 4.6 X 10(-6) M. Time of incubation and enzyme concentration studies were conducted. The analysis of retinal HIOMT activity in groups of trout killed over a day--night period did not detect a rhythm in enzyme activity. Results of these investigations are discussed in terms of the evolution of melatonin synthesis and functions.

Nonpineal melatonin in the alligator (Alligator mississippiensis).

All living and most fossil representatives of the reptilian subclass Archosauria lack pineal bodies. Arrhythmic, low-level, nonpineal melatonin is present, however, in the blood of Alligator mississippiensis. Although pineal bodies have been implicated in circadian phenomena, these results suggest that arrhytmic melatonin in alligators may not be involved incircadian events and indicate that the pineal is not the only source of the hormone melatonin. The evolutionary loss of the pineal in Archosauria occurred during the Mesozoic, and era noted for its seasonal stability. Arrhythmic melatonin titers inalligators and pineal loss in alligators and other archosaurs may be related to Mesozoic seasonal stability.

Melatonin synthesis by the retina.

Melatonin fulfills many of the criteria for classification as a hormone. Until recently it was considered to be elaborated exclusively by pineal organs. Melatonin synthesis by other tissues has been indicated but not demonstrated unequivocally. Trout retinas in a whole-organ culture system in vitro synthesized tritiated melatonin from a substrate containing tritiated serotonin. This raises the possibility that the trout retina is an endocrine organ.

Thyroxine-induced activation of hypothalamo-hypophysial axis in neotenic salamander larvae.

Thyroxine injected into the hypothalamus of neotenic Ambystoma tigrinum induces metamorphosis by activating hypothalamo-hypophysial stimulatory control of thyroid activity, thereby removing the hypothalamic block to metamorphosis.