Porphyrinic dyads and triads assembled around iridium(III) bis-terpyridine: photoinduced electron transfer processes.

Multicomponent arrays based on a central iridium(III) bis-terpyridine complex (Ir) used as assembling metal and free-base, zinc(II) or gold(III) tetraaryl-porphyrins (PH(2), PZn, PAu) have been designed to generate intramolecular photoinduced charge separation. The rigid dyads PH(2)-Ir, PZn-Ir, PAu-Ir, and the rigid and linear triads PH(2)-Ir-PAu, PZn-Ir-PAu, as well as the individual components Ir, PH(2), PZn, PAu have been synthesized and characterized by various techniques including electrochemistry. Their photophysical properties either in acetonitrile or in dichloromethane and toluene have been determined by steady-state and time-resolved methods. In acetonitrile, excitation of the triad PH(2)-Ir-PAu leads to a charge separation with an efficiency of 0.5 and a resulting charge-separated (CS) state with a lifetime of 3.5 ns. A low-lying triplet localized on PH(2) and the presence of the heavy Ir(III) ion offer the CS state an alternative deactivation path through the triplet state. The behavior of the triad PZn-Ir-PAu in dichloromethane is rather different from that of PH(2)-Ir-PAu in acetonitrile since the primary electron transfer to yield PZn(+)()-Ir(-)-PAu is not followed by a secondary electron transfer. In this solvent, both unfavorable thermodynamic and electronic parameters contribute to the inefficiency of the second electron-transfer reaction. In contrast, in toluene solutions, the triad PZn-Ir-PAu attains a CS state with a unitary yield and a lifetime of 450 ns. These differences can be understood in terms of ground-state charge-transfer interactions as well as different stabilization of the intermediate and final CS states by solvent.

Shuttles and muscles: linear molecular machines based on transition metals.

Transition-metal-containing rotaxanes can behave as linear motors at the molecular level. The molecules are set into motion either by an electrochemical reaction or using a chemical signal. In a first example, a simple rotaxane is described that consists of a ring threaded by a two-coordination-site axle. The ring contains a bidentate ligand, coordinated to a copper center. The axle incorporates both a bidentate and a terdentate ligand. By oxidizing or reducing the copper center to Cu(II) or Cu(I) respectively, the ring glides from a given position on the axle to another position and vice versa. By generalizing the concept to a rotaxane dimer, whose synthesis involves a quantitative double-threading reaction triggered by copper(I) complexation, a molecular assembly reminiscent of a muscle is constructed. By exchanging the two metal centers of the complex (copper(I)/zinc(II)), a large-amplitude movement is generated, which corresponds to a contraction/stretching process. The copper(I)-containing rotaxane dimer is in a stretched situation (overall length approximately 8 nm), whereas the zinc(II) complexed compound is contracted (length approximately 6.5 nm). The stretching/contraction process is reversible and it is hoped that, in the future, other types of signals can be used (electrochemical or light pulse) to trigger the motion.

Photoreceptor cells of the pike pineal organ as cellular circadian oscillators.

In the pike pineal, the rhythm of melatonin (MEL) secretion is driven by a population of cellular circadian oscillators, synchronized by the 24 h light/dark (LD) cycle. Because the pineal photoreceptor contains both the input and output pathways of the clock, this cell is likely to be a cellular circadian system by itself. To support this idea, we have dissociated and cultured pike pineal cells as well as purified photoreceptors. In culture, the pineal cells reassociated in follicles, surrounded by collagen fibres. At the electron microscopic level, they appeared well preserved. Total cells consisted mainly of photoreceptors and glia. Purified cells corresponded exclusively to photoreceptors. Under LD, MEL production was rhythmic. Under constant darkness (DD), the rhythm was well sustained for at least six 24 h cycles (tau = 24/27 h) with 1 x 10(6) total cells/well or below; with 2 x 10(6) total cells/well, a strong damping occurred towards high levels as soon as after the second cycle. At the density of 0.5 x 10(6) cells/well, purified photoreceptors produced less MEL than an equivalent amount of total cells. However, the pattern of the oscillations was similar to that observed with 2 x 10(6) total cells, i.e. a damping occurred rapidly. Decreasing the density to 0.125 x 10(6) photoreceptors/well resulted in a loss of homogeneity among replicates. The production of melatonin by single photoreceptors was monitored by means of the reverse haemolytic plaque assay. Both under LD and under DD, the number of photoreceptors releasing melatonin was higher during the (subjective) dark than during the (subjective) light. The results provide strong support to the idea that the pike pineal photoreceptor is a cellular circadian system. Expression of the oscillations seemed to depend on several factors, including cell to cell contacts between photoreceptors. There is indication that also MEL and glia might be involved.

Circadian regulation of hydroxyindole-O-methyltransferase mRNA in the chicken pineal gland in vivo and in vitro.

The production of the pineal hormone melatonin displays circadian variations with high levels at night. The last enzyme involved in melatonin biosynthesis is hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The expression of the mRNA encoding chicken HIOMT was investigated in vivo and in vitro throughout the light/dark cycle, in constant darkness and with light interruption of the dark phase. The stability of HIOMT mRNA was also examined. A day/night rhythm of HIOMT mRNA levels, with a peak at the midlight phase, was observed in vivo as well as in vitro. Constant darkness did not abolish this rhythm in vivo. One cycle of the HIOMT mRNA rhythm could be observed in constant darkness in vitro. In addition, a stimulatory effect of light on HIOMT mRNA levels during the dark phase could be observed in vivo as well as in vitro. HIOMT mRNA stability was not affected by light or dark conditions, as demonstrated by chase experiments with actinomycin D. The results indicate that the daily changes in HIOMT mRNA concentration reflect transcriptional regulation by circadian oscillators and photosensory mechanisms that are endogenous to the pineal gland.

Partial characterization of serotonin N - acetyltransferases from northern pike (Esox lucius, L.) pineal organ and retina: effects of temperature.

In vertebrates, the nocturnal rise in pineal organ and retinal melatonin synthesis results from the increase in the activity of the serotonin N-acetyltransferase (NAT), a cAMP-dependent enzyme. In the fish pineal organ in culture, light and temperature act in a similar manner on cAMP content and NAT activity. It is not known whether the effects of temperature are mediated through cAMP or through modifications of NAT kinetics. The present study was designed: (1) to find out whether NAT activity from pineal organ homogenates is similar to NAT activity from pineal organs in culture, with regard to variations in temperature, and (2) to compare NAT activity from the pineal organ and the retina. Pineal organ and retinal NAT activity increased linearly with protein concentrations. Higher activities were obtained with 0.2 mol/l of phosphate buffer, pH 6. Higher molarity or a higher pH induced a decrease in retinal and pineal organ NAT activity: retinal NAT was more sensitive than pineal organ NAT to changes in molarity, whereas the opposite held true as far as pH was concerned. Pineal organ and retinal NAT obeyed the Michaelis-Menten equation with respect to increasing concentrations of acetyl-coenzyme A. With increasing concentrations of tryptamine: (1) pineal organ NAT activity increased in a manner suggesting positive co-operativity, (2) retinal NAT displayed, after an initial increase, inhibition by substrate. The kinetics of the reactions were temperature dependent. Maximal activities were reached at 18/20 degrees C in the pineal organ and at 37 degrees C in the retina. The present study is the first to describe the optimum conditions for the assay of NAT activity in homogenates from the retina of fish and from the pineal organ of poikilotherms, and also the first to compare some characteristics of NAT activity from these two analogous organs. Our results suggest that the effects of temperature on melatonin production are mediated, at least in part, through modifications of NAT kinetics. Future studies will aim to clarify whether the activities measured in the pineal organ and retinal homogenates reflect the presence of one or of several enzymes.

Hydroxyindole-O-methyltransferase in the chicken retina: immunocytochemical localization and daily rhythm of mRNA.

In the vertebrate retina and pineal gland, melatonin production displays diurnal variations with high levels at night. Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) catalyses the last step of melatonin biosynthesis. In the present study, a cDNA encoding chicken HIOMT was used to examine the effects of environmental lighting on HIOMT mRNA expression in the chicken retina. A day/night rhythm of HIOMT mRNA level was observed, with an average 5-fold increase during the night. Light strongly suppressed the night-time rise in HIOMT mRNA concentration while darkness prevented its daytime fall. An antibody directed against chicken HIOMT was used for immunocytochemical identification of retinal melatoninergic cells. HIOMT immunoreactivity could be observed in rods as well as in cones. However, the lowest levels of HIOMT immunoreactivity were always observed in the accessory cones of double cones. A few HIOMT-positive cell bodies could also be observed in the inner nuclear layer. Altogether, these data indicate that HIOMT gene expression in the retina is organized on a daily basis as a direct response to light, and that the different types of photoreceptors may not be equally involved in melatonin production.

Age-related changes in 2-[(125)I]-iodomelatonin binding sites in the brain of sea breams (Sparus aurata, L.).

The pineal organ of fish, through its 24h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g., reproduction, growth). The target sites for melatonin are poorly known in fish, especially marine species. A radioligand study was undertaken using the gilthead sea bream (Sparus aurata) maintained under natural temperature and photoperiod (at 28°N latitude). This species exhibits the property of changing sex during growth. Brains of one year-old males were collected at 16:00h and brains of three year-old females at 03:00, 10:00, 16:00 and 23:00h. Membrane homogenate receptor assays were run using 2-[(125)I]iodomelatonin as a ligand. Binding sites were detected in brains of young and old fish. In the younger, the exhibited a Bmax between 3.52 and 4.29 fmol mg protein(-1) and a KD between 358-380 pmol l(-1). In the older fish, the KD varied according to a daily pattern: values were three times higher at 03:00 and 10:00h (500-600 pmol l(-1)) than at 16:00 and 23:00h (150-300 pmol l(-1)). The number of sites also were higher at 03:00 and 10:00h (180-200 fmol mg protein(-1)) than at 16:00 and 23:00h (95-110 fmol mg protein(-1)). Melatonin and iodomelatonin displaced 2-[(125)I]iodomelatonin binding in a dose dependent manner, the second being more potent than the first. Binding was also inhibited by GTP. The results provide the first evidence for the presence of membrane melatonin binding sites in the brain of an exclusively marine fish. They suggest that their number and affinity varies during growth and throughout a light/dark cycle. Future experiments will aim to precise the anatomical location and role of these binding sites.

Hydroxyindole-O-methyltransferase gene expression in the pineal gland of chicken embryo: development of messenger RNA levels and regulation by serum.

Hydroxyindole-O-methyltransferase (HIOMT), the enzyme which catalyzes the final step of melatonin biosynthesis, constitutes a marker of the functional differentiation of pineal cells. In addition, a day/night rhythm of HIOMT mRNA concentration, previously described in the chicken pineal gland [6], would suggest that HIOMT gene transcription is one output of the circadian system that controls pineal function. The study sought to monitor the developmental expression of HIOMT mRNA in the chick pineal gland and to investigate a possible role of instructive signals in this differentiation process. RT-PCR analysis indicated that HIOMT mRNA is expressed at embryonic day 8 (E8). At E12, HIOMT mRNA became detectable on northern blots and traces of HIOMT activity could be measured. HIOMT mRNA concentration increased 100-fold between E14 and day 10 post-hatch, then levelled off. A day/night rhythm of HIOMT mRNA concentration was readily observed in the pineal gland of 2-day-old chicks. Pineal glands isolated on minimum culture medium at E11 stopped developing HIOMT gene expression. However, the addition of serum to the culture medium restored HIOMT mRNA concentration to the levels observed in vivo. The data suggest that the functional differentiation of melatoninergic cells observed during the second week of embryonic life may be controlled [correction of controled] by serum factors.

Adenosine A2 receptor-mediated stimulation of cyclic AMP in cultured chicken pineal cells.

The pineal gland of vertebrates produces the time-keeping hormone melatonin in a rhythmic manner. Regulation of melatonin production is a multifactorial process. In the chicken, light, perceived through the skull, and norepinephrine, acting through alpha 2-adrenergic receptors, synergistically inhibit day time melatonin production. In addition, adenosine exerts autocrine/paracrine modulatory effects on melatonin secretion. In an attempt to elucidate how these effects of adenosine are mediated, chicken pineal cells were cultured, in the dark during day time, in the presence of different analogs of adenosine. When the adenosine transmembranous carrier was inhibited, chloroadenosine stimulated cyclic AMP (cAMP) accumulation in a time- and dose-dependent manner. The effects were antagonized by 8-phenyltheophylline, an antagonist at the A1/A2 adenosine receptors. A dose-dependent stimulation of cAMP accumulation was also obtained with other adenosine agonists, with the following order of potency: N-ethylcarboxamidoadenosine > cyclopentyladenosine > R-phenyl-isopropylade-nosine. The stimulatory effect of the latter compound was still observed when basal cAMP levels were increased in the presence of forskolin. Under our experimental conditions no inhibition of cAMP content was observed. Our results are consistent with the idea that stimulation of melatonin secretion by adenosine analogs is mediated through A2 receptors.

Rhythmic secretion of melatonin by the superfused pike pineal organ: thermo- and photoperiod interaction.

In the pineal organ of the pike (Esox lucius, teleost), the rhythmic production of melatonin by the photoreceptor cells is governed by a population of endogenous circadian oscillators, synchronized and entrained by the light/dark (L/D) cycle. Production of melatonin is inhibited by light and highly increased in the dark. In the present study, isolated and superfused pike pineals were exposed to a 24-hour temperature cycle of high (10 degrees C) or low (4 degrees C) amplitude, either under L/D, or under constant darkness. Under L/D, photoperiod is the dominant entraining stimulus for the melatonin secretion rhythm. It was high during the dark phase and low during the light phase, either under cold light/warm dark or under warm light/cold dark conditions. Under a warm light/cold dark cycle, the amplitude of the melatonin rhythm was reduced. In pineals cultured at 10 degrees C, a 20 degrees C temperature pulse potentiated or reduced the night-induced rise in melatonin production when applied, during the dark or during the preceding light phase, respectively. Under constant darkness, high- or low-amplitude temperature cycles could synchronize the rhythmic production of melatonin, which peaked with the high temperature. However, the shape of the oscillation could appear modified under warm subjective-L/cold subjective-D, depending on the experimental conditions. Finally, the rhythmic release of melatonin synchronized by a temperature cycle under constant darkness was no longer observed after removal of the external zeitgeber. The present study provides direct evidence that the pineal acts as a photothermotransducer.

Calciproteins regulate cyclic AMP content and melatonin secretion in trout pineal photoreceptors.

Photoreceptor cells of the fish pineal transduce photoperiodic information into the rhythmic secretion of melatonin. The nocturnal rise in melatonin secretion has been associated with an increase in cyclic AMP (cAMP) production and with an entry of Ca2+ ions through L-type voltage-dependent channels. It is shown here that two inhibitors of calciproteins, W7 and calmidazolium, inhibit melatonin secretion and, to a lesser extent, cAMP levels in cultured trout pineal photoreceptors. Kinetic studies indicated that melatonin secretion was affected earlier than cAMP in cells cultured in the presence of W7. The present results provide evidence that Ca2+ acts through one or more calciproteins to regulate melatonin production. It is suggested that Ca2+/calciprotein complexes might act at two different sites, one involving regulation of cAMP metabolism, and the other being independent from cAMP.

Calcium and melatonin production in dissociated trout pineal photoreceptor cells in culture.

Trout pineal cells maintained in primary culture produce melatonin in high amounts during night time and low amounts during daytime. The dark-induced increase in melatonin production was enhanced, in a dose-dependent manner, by elevating extracellular calcium concentration. Low external calcium concentration reduced nocturnal and diurnal melatonin production. Bay K 8644 increased, in a dose-dependent manner, the dark-induced rise in melatonin output, and this effect was antagonized by nifedipine and verapamil. This suggests a role for the dihydropyridine calcium channels in the regulation of the melatonin output. To confirm this, patch-clamp recordings (whole-cell perforated) were run in a 20 mmol/l barium medium at different holding potentials from -80 mV. A voltage-dependent inward current was activated from -30 mV to +40 mV with a maximal amplitude being observed at 0 mV. This current was drastically increased in the presence of Bay K 8644. Nifedipine inhibited the current both in the absence or in the presence of Bay K 8644. Our results are consistent with the idea that extracellular calcium participates in the control of melatonin secretion by photoreceptor cells. It is suggested that activation of the voltage-dependent L-type channel may modulate this secretion.

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

The fish pineal organ contains typical and, in some species, modified photoreceptor cells involved in the photoperiodic control of melatonin production. In the majority of species studied, the rhythm in melatonin production is driven by an intra-pineal circadian oscillator synchronized by the light:dark cycle. In the present study, it is shown that the endogenous rhythm in melatonin release of superfused pike pineals maintained under constant darkness is expressed at temperatures of 19 degrees C, 20 degrees C, 25 degrees C, and 30 degrees C (period > 24 hr), but not at temperatures of 10 degrees C and 15 degrees C. Under constant darkness, pineal fractions containing either typical photoreceptors, modified photoreceptors, or both behaved like total organs. Dissociated pike pineal cells, cultured statically at 20 degrees C, expressed a high amplitude rhythm in melatonin secretion under a light:dark cycle. Under constant darkness, circadian oscillations, which appeared better sustained than in organ culture, were also observed. This study provides the first evidence that the rhythmic production of melatonin, by a fish pineal, is driven by a population of circadian oscillators or clocks. It is hypothesized that each typical and modified photoreceptor might be the locus of a circadian clock. Damping of the overall rhythm under constant darkness might reflect the desynchronization (uncoupling) between these clocks and/or damping of individual oscillators.

Immunocytochemical localisation of hydroxyindole-O-methyltransferase in pineal photoreceptor cells of several fish species.

Melatonin is an internal "Zeitgeber," involved in the timing and control of a number of rhythmic functions and behaviours. Its synthesising cells remain to be identified in the fish pineal. The last step in the melatonin biosynthetic pathway is catalysed by the enzyme hydroxyindole-O-methyltransferase. An affinity-purified antibody, directed against chicken pineal hydroxyindole-O-methyltransferase, was used in the present study to identify the melatonin synthesising cells in four fish species: a primitive chondrostean (sturgeon), a saltwater teleost (dorado), and two freshwater teleosts (pike, trout). Western blot immunolabeling of pike and trout pineal proteins revealed a single band at 38 KDa, which corresponds to the known molecular weight of the enzyme in bovine, rat, and chicken pineal. Regardless of the species, a specific immunocytochemical labeling, visualised by means of the peroxidase-antiperoxidase method, was exclusively associated with the photoreceptor cells. These results provide evidence that photoreceptors of the fish pineal are responsible for the biosynthesis of 5-methoxyindoles, including melatonin. In the pike, reactions were less intense in the distal portion of the pineal vesicle than in the other regions of the organ. It is questioned whether this might be related to the existence of a germinative zone, generating new photoreceptor cells in this distal portion. Hydroxyindole-O-methyltransferase has been previously demonstrated in mammalian pinealocytes, and modified photoreceptors of the avian pineal. It is now demonstrated in pineal photoreceptors of a primitive fish and of more evolved saltwater and freshwater fish. The results strengthen the view that these cells are related through phylogeny and that their well conserved melatoninergic function appears early in the course of evolution.

[The sensory potential of the ring finger. The value of electromyographic diagnosis of carpal tunnel syndrome]. / Potentiel sensitif de l'annulaire. Intérêt dans le diagnostic électromyographique du syndrome du canal carpien.

Accurate diagnosis of carpal tunnel syndrome relies on electromyography. The usual criteria (sensory conduction velocity and motor latency) fail to identify 10% of cases of carpal tunnel syndrome. The sensory innervation of the ring finger (digit IV) is shared by the median and ulnar nerves. This anatomic feature is the basis of a novel electromyographic test: measurement of the sensory potential of digit IV by an orthodromic method with stimulation of the finger via a ring electrode and recording at the wrist. Analysis of the sensory potential of digit IV in 200 subjects with no median nerve compression afforded an invariably synchronised trace with no error of interpretation since it required no additional measurements. A study of 179 cases in 124 patients suspected of carpal tunnel syndrome showed pathological traces that could be graded into five levels of severity. Comparison with sensory conduction velocity and distal motor latency confirmed its validity and especially its usefulness in mild forms. The sensitivity of the test is excellent since false negatives are theoretically due to anomalies in the sensory innervation of digit IV, the reported occurrence of which varies widely (about 15%). The were no false positives. Its reproducibility, painlessness and sensitivity have led us to make this test compulsory in all patients suspected of CTS in our practice.

Regulation of hydroxyindole-O-methyltransferase gene expression in the pineal gland and retina.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step of melatonin biosynthesis and appears to be specifically expressed in the pineal gland and in the retina. This review deals with the regulation of HIOMT by environmental light and with the developmental aspects of HIOMT expression in chicken and rat. Early studies based on HIOMT activity measurements and more recent studies involving cDNA hybridization to HIOMT mRNA are taken into consideration. Together, the data reveal that long term regulation of HIOMT by light would rely on a day/night rhythm of HIOMT gene transcription, coupled to a slow turnover of the protein. Rapid changes in HIOMT mRNA levels and early expression during embryonic development suggest that further studies on this gene may shed light on the molecular mechanisms involved in the differentiation of the melatoninergic function and in its regulation by light, both in the pineal gland and in the retina.

Detection of estrogen receptor mRNA in trout pineal and retina: estradiol-17 beta modulates melatonin production by cultured pineal photoreceptor cells.

Pineal photoreceptor cells produce the neurohormone melatonin, a major "Zeitgeber" of the organism. This compound is involved in the control of development, growth, sexual maturation, and seasonal reproductive cycles. The present study reports that the photosensitive pineal organ and the retina of the trout express a 3.5-kb mRNA corresponding to the estradiol-17 beta receptor. The effects of estradiol-17 beta on melatonin production by cultured pineal photoreceptor cells were also studied. Under a light/dark (LD:12/12) cycle, these cells maintained a rhythmic secretion of melatonin, with higher amounts being released during the dark phase. The amplitude of the rhythm tended to increase with time in culture. Application of estradiol-17 beta during the dark phase of an LD cycle (i.e., for 12 hr) affected melatonin release in a dose-dependent manner; low concentrations (10(-10) to 10(-8) mol/liter) were inhibitory, whereas high concentrations (over 10(-7) mol/liter) were stimulatory (when compared to the values obtained at 10(-9) mol/liter). When estradiol-17 beta was given continuously for several 24-hr LD cycles, the inhibitory effect observed during the first dark phase disappeared in subsequent dark periods. In the presence of estradiol-17 beta, at concentrations ranging from 10(-10) to 10(-5) mol/liter, a high amplitude rhythm in melatonin secretion returned more rapidly than in controls. The present results suggest that estradiol-17 beta receptors are expressed in the fish pineal and retina and that estradiol-17 beta modulates melatonin secretion by cultured pineal photoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

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).

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.

Cellular localization of hydroxyindole-O-methyltransferase mRNA in the chicken pineal gland.

Hydroxyindole-O-methyltransferase (HIOMT), is the last enzyme of the melatonin biosynthesis pathway. We have applied immunocytochemistry and in situ hybridization to localize HIOMT producing cells, in the chicken epithalamus including the pineal gland. The selectivity of the hybridization signal over melatoninergic cells was ascertained by comparison with the immunolabelling. HIOMT transcripts could be observed in the two cell layers of the pineal follicles and in the pineal stalk, but not in other epithalamic regions. These results indicate that pineal modified photoreceptors and parafollicular pinealocyte-like cells represent the two populations of melatonin-producing cells of the chicken epithalamus.