Melatonin modulates secretion of growth hormone and prolactin by trout pituitary glands and cells in culture.

In Teleost fish, development, growth, and reproduction are influenced by the daily and seasonal variations of photoperiod and temperature. Early in vivo studies indicated the pineal gland mediates the effects of these external factors, most probably through the rhythmic production of melatonin. The present investigation was aimed at determining whether melatonin acts directly on the pituitary to control GH and prolactin (PRL) secretion in rainbow trout. We show that 2-[125I]-iodomelatonin, a melatonin analog, binds selectively to membrane preparations and tissue sections from trout pituitaries. The affinity was within the range of that found for the binding to brain microsomal preparations, but the number of binding sites was 20-fold less than in the brain. In culture, melatonin inhibited pituitary cAMP accumulation induced by forskolin, the adenyl cyclase stimulator. Forskolin also induced an increase in GH release, which was reduced in the presence of picomolar concentrations of melatonin. At higher concentrations, the effects of melatonin became stimulatory. In the absence of forskolin, melatonin induced a dose-dependent increase in GH release, and a dose-dependent decrease in PRL release. Melatonin effects were abolished upon addition of luzindole, a melatonin antagonist. Our results provide the first evidence that melatonin modulates GH and PRL secretion in Teleost fish pituitary. Melatonin effects on GH have never been reported in any vertebrate before. The effects result from a direct action of melatonin on pituitary cells. The complexity of the observed responses suggests several types of melatonin receptors might be involved.

14-3-3 proteins in pineal photoneuroendocrine transduction: how many roles?

Recent studies suggest that a common theme links the diverse elements of pineal photoneuroendocrine transduction--regulation via binding to 14-3-3 proteins. The elements include photoreception, neurotransmission, signal transduction and the synthesis of melatonin from tryptophan. We review general aspects of 14-3-3 proteins and their biological function as binding partners, and also focus on their roles in pineal photoneuroendocrine transduction.

Melatonin receptors in the pituitary of a teleost fish: mRNA expression, 2-[(125)I]iodomelatonin binding and cyclic AMP response.

Melatonin has for a long time been involved in the photoperiodic control of fish physiology (growth, reproduction) and behavior (locomotor activity); but its mechanisms of action are not understood. We show here that 2-[(125)I]iodomelatonin binds specifically to membrane preparations from Pike (Esox lucius, L.) pituitaries (K(D): 556 pM; B(max): 2.8 fmol/mg proteins). Radioautography indicated that the binding was restricted to a part of the pituitary only. Using polymerase chain reaction from pike genomic DNA, we subcloned two partial cDNAs encoding the P1.4 (Mel(1a)) and the P2.6 (Mel(1b)) melatonin receptor subtypes. The two corresponding transcripts were expressed in the pituitary as revealed by RT-PCR assay and Southern blot hybridization. In culture, melatonin inhibited in a time- and dose-dependent manner cyclic AMP levels in pituitaries cultured in the presence of forskolin, an adenylyl cyclase activator. This is the first evidence for the expression of melatonin receptors and binding sites, and for the modulation of a second messenger by melatonin in the pituitary of a nonmammalian species. We suggest that in fish, the melatonin-mediated photoperiodic control of neuroendocrine functions involves, at least, a direct effect on the pituitary.

Expression of melatonin receptors and 2-[125I]iodomelatonin binding sites in the pituitary of a teleost fish.

The mechanisms of the photoperiodic control of fish physiology (growth, reproduction) and behavior (locomotor activity) are far from being understood. We show here that 2-[125I]iodomelatonin binds specifically to membrane preparations from pike (Esox lucius, L.) pituitaries (KD: 556 pM; Bmax: 2.8 fmol/mg proteins). Radioautography indicated that the binding was restricted to a part of the pituitary only. Using polymerase chain reaction from pike genomic DNA we were able to isolate, subclone and sequence two fragments. The so-called P4 and P8 fragments displayed homology with, respectively, the Mel1a and Mel1b receptor subtypes. The P4 and P8, probes allowed detection of mRNAs corresponding to these receptors in different areas of the brain, including the pituitary. This is the first evidence that melatonin receptors and binding sites are expressed in the pituitary of a non-mammalian species. We suggest that in fish the melatonin-mediated photoperiodic control of neuroendocrine functions might involve a direct effect on the pituitary.

Daily and circadian variations in 2-[125I]-iodomelatonin binding sites in the pike brain (Esox lucius).

The fish pineal organ, through its 24 h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g. reproduction, growth). We have investigated the binding of 2-[125I]iodomelatonin to whole brain membrane preparations from pikes (Esox lucius L., teleost) maintained for 24-48 h under different photoperiodic conditions. Specific binding was stable, reversible, saturable and sensitive to the presence of a GTP analogue. Scatchard analysis revealed one class of binding sites. Displacement experiments suggested the presence of two components with affinities in the femtomolar and nanomolar range of concentrations, respectively. The Bmax exhibited monophasic nycthemeral variations, with higher values at the light-to-dark transition (34.0 +/- 4.5 fmol/mg protein) and low values during the second half of night (10.0 +/- 1.0 fmol/mg protein). Under the same conditions, the KD exhibited biphasic variations: values were low during daytime and at the middle of the dark phase (approximately 100 pM); they were high at the beginning (approximately 225 pM) and at the end (approximately 330 pM) of the night. These variations were maintained under constant light (LL) and constant darkness (DD). Thus, the variations in the number and affinity of the melatonin binding sites were controlled by circadian oscillators, synchronized by the photoperiod. The nature of these oscillators is not known. Therefore, in fish, we suggest that the photodependent effects of melatonin result from the circadian variations of both its production by the pineal and its binding sites in the brain.

Variations in cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate content and efflux from the photosensitive pineal organ of the pike in culture.

The photoreceptor cells of the pike pineal organ transduce 24-h light/dark (LD) information to synchronize the clocks driving the melatonin (MEL) rhythm. In fish, the nocturnal rise in MEL synthesis is associated with an increase in cyclic adenosine 3', 5'-monophosphate (cAMP) production and with Ca2+ entry, through voltage-gated channels. Light induces inhibition of MEL synthesis and a depression of cAMP content, as well as closure of Ca2+ channels. Cyclic guanosine 3',5'-monophosphate (GMP) levels also are reduced upon acute illumination but this second messenger of phototransduction does not appear to be directly involved in the control of MEL metabolism. It is not known whether cAMP and/or cGMP are components of the clock machinery. In this study we measured cAMP and cGMP contents (static culture) and release (perifusion culture) using pike pineal organs maintained under LD or DD (constant darkness). Under LD, cAMP levels were low at noon and midnight, and high at dawn and dusk, in organs as well as in perfusates. This pattern was maintained under DD, with a major peak occurring at the beginning of subjective light, and a minor peak at the beginning of subjective darkness; only one peak during the subjective light was seen in the perfusates. Under DD, the MEL rhythm displays only one peak during the subjective night. It is suggested that increases in cAMP might not always be correlated with increases in MEL secretion. Under LD, variations in cGMP content were not statistically significant; however, in the perfusates, the levels were higher during the night than during the day. This suggests that: (1) extrusion participates in the regulation of intracellular levels of cGMP, (2) nocturnal synthesis of cGMP is higher than its catabolism, and (3) synthesis is increased during the day to compensate for the light-induced activation of catabolism. Under DD, the cGMP content and release were higher during the subjective night than during the subjective day, revealing a circadian component in the regulation of cGMP metabolism. This may provide the basis for the generation of membrane-related circadian events including variations in membrane potential, in the opening/closure of voltage-gated channels (e.g. Ca2+ channels), or in enzyme activities (adenylyl cyclase, cGMP-dependent phosphodiesterase).