Jean-Pierre Collin

Regulation of the Rhythmic Melatonin Secretion by Fish Pineal Photoreceptor Cells

Many, if not most, biochemical, physiological and behavioural events are rhythmic. Rhythms with a periodicity of 24 h (daily) or one year (annual) represent major components in the adaptation of organisms to their environment. Environmental factors, such as light and temperature, play an important role in the synchronization of these rhythms. In vertebrates, synchronization is mediated through the circadian system, which is composed of sensors and circadian oscillators. These include the pineal organ, the lateral eyes and the suprachiasmatic nuclei of the hypothalamus.

Rhythmic Melatonin Biosynthesis in a Photoreceptive Pineal Organ: A Study in the Pike

Activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase, involved in the biosynthesis of melatonin from serotonin, were assessed over 24 h in the pineal organ of pikes (Esox lucius, L.; teleosts) entrained to natural (winter) environmental conditions. Only NAT activity exhibited daily changes, rising at the onset of darkness and resuming low values shortly before the end of the scotophase. The rhythm was damped under constant darkness, lower and higher values being shifted towards intermediate ones. NAT activity was lowered under constant light; however, a significant increase was seen at the end of the subjective night, suggesting the existence of a low-amplitude rhythm. Illumination of the pikes at the middle of the scotophase induced inactivation of roughly half of the maximal NAT activity, possibly indicating the existence of one photolabile and one photostable enzymatic component. Under natural conditions, radioassayable melatonin of the pike pineal organ displayed daily variations which paralleled those of NAT activity. Melatonin production thus appears to reflect the daily changes of NAT, synchronized to the light/dark cycle.

Regulation of Melatonin Production by Pineal Photoreceptor Cells: Role of Cyclic Nucleotides in the Trout (Oncorhynchus mykiss)

Abstract: 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.

Multiple circadian oscillators in the photosensitive pike pineal gland: A study using organ and cell culture

Bolliet V, Bégay V, Ravault J‐P, Ali MA, Collin J‐P, Falcón J. Multiple circadian oscillators in the photosensitive pike pineal gland: A study using organ and cell culture. J. Pineal Res. 1994: 16: 77–84.

Characterization of melatonin binding sites in chicken and human intestines

The radioligand 2-[125I]iodomelatonin was used to study melatonin binding sites in chicken and human intestines. In the chicken duodenum, 2-[125I]iodomelatonin binding sites were enriched in the musculosa layer (Bmax approximately 1 fmol/mg protein) as compared to the mucosa/submucosa layer (Bmax approximately 0.2 fmol/mg protein). 2-[125I]iodomelatonin bound with a Kd of 68 +/- 18 pM (mean +/- S.E.M., n = 13) and was displaced by melatonin with a Ki of 0.3 nM. The Kd value for 2-[125I]iodomelatonin was increased 2- to 4-fold by a GTP analog, suggesting that the binding sites might be coupled to a G-protein. The affinity order of nine melatonin analogs at the enteric binding sites was in agreement with the pharmacological profile of melatonin receptors described in other tissues. In the human jejunum, 2-[125I]iodomelatonin binding could be observed in the mucosa/submucosa layer (Kd = 150-200 pM, Bmax = 0.7 fmol/mg protein). The radioligand was efficiently displaced by melatonin (Ki = 0.6 nM) but only marginally by N-acetyltryptamine (Ki = 22 microM) and serotonin (Ki = 14 microM).

In Vitro Uptake and Metabolism of [3H]Indole Compounds in the Pineal Organ of the Pike. I. A Radiochromatographic Study

Thin layer chromatography analysis of [3H]serotonin and [3H]melatonin metabolites synthetized in vitro by the pineal organ of the pike was performed. After a 10‐min pulse, [3H]serotonin was mainly converted into [3H]‐5‐hydroxyindoleacetic acid (37%), [3H]‐5‐hydroxytryptophan and [3H]‐5‐methoxytryptophan (12 to 14%), and [3H]‐5‐hydroxytryptophol and [3H]‐5‐methoxytryptophol (3.5 and 9%) at the onset of darkness. When the pulse was followed by postincubations (in a cold medium) of increasing duration (15, 30, and 60 min), it appeared that 1) the amount of [3H]‐5‐hydroxyindoleacetic acid decreased, 2) that of [3H]‐5‐hydroxytryptophol decreased faster than that of [3H]‐5‐methoxytryptophol, and 3) the amounts of [3H]‐5‐hydroxy‐ and [3H]‐5‐methoxytryptophan increased. [3H]‐N‐acetylserotonin, [3H]melatonin, and [3H]‐5‐methoxytryptamine were found in very low amounts.

Pineal-retinal molecular relationships; immunocytochemical evidence of calbindin-27 kDa in pineal transducers

Calbindin-27 kDa immunocytochemical localization was studied concurrently in the pineal organ and retina from human as well as representatives of all vertebrate classes. Calbindin immunoreactivity was demonstrated in retinal cones (but not in rods) and in pineal transducers (cone-like and modified photoreceptor cells, pinealocytes) of a majority of amniotes. In contrast, no labelling was observed in anamniotes, except in retinal cones of the toad. Labelling was distributed through all cellular compartments (outer and inner segments, perikarya, pedicles or processes) of pineal transducers and retinal cones. Intra- and interspecific variations of calbindin contents are discussed.

5'-Nucleotidase activity in the pineal organ of the pike

SummaryTo date, it is still unknown whether the metabolism of purine nucleotides and nucleosides plays an important role in the pineal organ of lower vertebrates. We have therefore investigated the sites of 5′-nucleotidase activity in the pineal organ of the pike (Esox lucius L.). Various ultracytochemical procedures were used. An intense ecto-5′-nucleotidase activity was characteristic of the entire plasma membrane of the phototransducers (cone-like and modified photoreceptor elements) and the interstitial cells, with exception of the portions facing the basal lamina of the pericapillary spaces. Additionally, intracellular sites of activity were also visualized in the inner segment and the pedicle of the phototransducers. Most of the intracellular deposits were apparently cytosolic and only few seemed to be associated with the membrane of the clear “synaptic” vesicles of the pedicle. Phagocytotic cells in the pineal lumen also showed a strong enzymatic activity on the outer surface of their plasmalemma (in ectoposition). This was apparently not the case for the cell types of the tissues surrounding the pineal vesicle. The present study emphasizes the importance of the occurrence and metabolism of purine nucleotides and nucleosides in a photoreceptive pineal organ.

Distribution of arrestin-like protein and β-subunit of GTP-binding proteins in quail choroid plexuses

Monoclonal antibodies (Mabs) directed against retinal arrestin (S-antigen) were used to detect and characterize this protein in choroid plexus (CP) of quails maintained during eight days, either under long-day photoperiods or in constant darkness. Immunocytochemistry and Western blotting confirmed the presence and the distribution of an arrestin-like protein in quail CP. Arrestin-like immunoreactivities in CP were compared with those obtained with Mabs to beta 36-subunit of G proteins (G beta), alpha-subunit of transducin and rhodopsin. Rhodopsin-like and transducin-like proteins could not be detected in choroidal cells, whereas intense positive reactions were observed with anti-G beta and anti-arrestin Mabs. The strongest immunoreactivities were found in choroidal ependymocytes of the lateral and IIIrd ventricles. In CP epithelial cells lining the IVth ventricle, very weak or no immunoreactivity could be detected with Mabs to arrestin, while Mab against G beta subunit always provided a positive reaction. In quails maintained in constant darkness, arrestin- and G beta-immunoreactivities of CP epithelial cells displayed changes in cellular distribution and intensity (decrease or disappearance of the immunoreactions). The strong arrestin-like immunoreaction located in the apical region of ependymocytes suggests the preferential association of the protein with choroidal microvilli and a possible role in cerebrospinal fluid production assumed by CP cells.