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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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)

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).

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.

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.

The indolic hormone melatonin is produced by the pineal gland according to a daily rhythm. The terminal step of melatonin synthesis is catalysed by hydroxyindole O-methyltransferase (HIOMT, EC 2.1.1.4). Adaptation to constant light or darkness modifies HIOMT activity and concentration. Using a cDNA probe encoding HIOMT, we investigated the effect of environmental lighting on HIOMT gene expression in the chicken pineal gland. HIOMT mRNA levels increased by 100% in constant light as compared with constant darkness. In addition, the present study disclosed the existence of a day/night rhythm of HIOMT gene transcription, with 3-fold higher mRNA levels at midday than at midnight. This transcriptional rhythm was not accompanied by day/night changes in HIOMT concentration, probably due to a slow turnover of this protein. Unexpected darkness did not prevent the daytime rise in HIOMT mRNA levels, whereas unexpected light prevented the night-time fall in HIOMT mRNA levels. Together, the data would suggest that the day/night rhythm of HIOMT gene transcription in the chicken pineal gland involves both a response to light and the activity of a biological oscillator.

The effects of prostaglandins (PGs) on the activity of the rate-limiting enzyme of melatonin biosynthesis, arylalkylamine-N-acetyltransferase (NAT) were investigated on primary cultures of dispersed chick pineal cells. In indomethacin-treated cells, PGs caused a four-fold increase in NAT activity. This response was associated with an eight-fold increase in cyclic AMP (cAMP) levels. The potency order of PGs was the same for NAT and for cAMP responses (PGE1 > PGE2 > PGF2 alpha >> cloprostenol). However, each PG tested was 30- to 200-fold more potent to increase NAT activity than to stimulate cAMP accumulation. As a result, half-maximal stimulation of NAT by PGs was not associated with an increase in cAMP levels. Half-maximal stimulation of NAT by PGE1 was highly sensitive to inhibition by a calcium/calmodulin antagonist (W-7). In contrast, maximal stimulation of NAT by PGE1 as well as stimulations evoked by either forskolin or 8-bromo-cAMP were poorly sensitive to inhibition by W-7. These results indicate that an increase in cAMP levels may be responsible for the maximal stimulation of NAT evoked by PGs, whereas half-maximal stimulation of NAT by PGs would rely principally on a calcium/calmodulin-dependent mechanism.

Pineal photoreceptor cells produce the neurohormone, melatonin, a major "Zeitgeber" of the organism. This compound has been involved in the control of development, growth, sexual maturation, and seasonal reproductive cycles. We investigated, here, the effects of estradiol-17 beta on melatonin production by cultured pineal photoreceptor cells. Under a light/dark (LD: 12/12) cycle, cultured trout pineal 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 spent in culture. Application of estradiol-17 beta during the dark phase of a LD cycle (i.e., for 12 h) affected melatonin release in a dose-dependent manner: low concentrations (10(-10) to 10(-8) mol/l) were inhibitory whereas high concentrations (over 10(-7) mol/l) were stimulatory. When estradiol-17 beta was applied continuously for several 24 h LD cycles, the inhibitory effect observed during the first dark phase disappeared later on. Rather, in the presence of estradiol-17 beta, at the concentrations of 10(-9) and 10(-6) mol/l, a high amplitude rhythm in melatonin secretion was recovered faster, when compared to controls. Replacement of 10% fetal calf serum by 0.1% bovine serum albumin did not affect the responses of the photoreceptor cells to 10(-6) mol/l of estradiol-17 beta. The present results bring the first evidence that estradiol-17 beta modulates melatonin secretion by cultured pineal photoreceptors. They further support the idea according to which sexual hormones exert a feedback regulation on the pineal. Pineal photoreceptors appear as multieffector cells which transduce information from, both, physical (photoperiod) and internal (chemical) factors.

The indolic hormone melatonin is produced by the pineal gland according to a daily rhythm. The terminal step of melatonin synthesis is catalyzed by hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). Adaptation to constant light or darkness modifies HIOMT activity and concentration. Using a cDNA probe encoding HIOMT, we investigated the effect of environmental lighting on HIOMT gene expression in the chicken pineal gland. HIOMT mRNA levels increased by 100% in constant light as compared with constant darkness. In addition, the present study disclosed the existence of a day/night rhythm of HIOMT gene transcription, with 3-fold higher mRNA levels at midday than at midnight. Unexpected darkness did not prevent the daytime rise in HIOMT mRNA levels, whereas unexpected light prevented the night-time fall in HIOMT mRNA levels. Together, the data would suggest that the day/night rhythm of HIOMT gene transcription in the chicken pineal gland involves both a response to light and the activity of a biological oscillator.

Trout pineal photoreceptor cells were dissociated by trypsin-DNase digestion and further purified by a Percoll gradient centrifugation. Total cells or purified photoreceptor cells were then embedded in a collagen gel, or layered on culture-treated polycarbonate membranes, or maintained in suspension, with RPMI 1640 medium or BGjb medium. It has been shown that cells maintain a rhythmic production of melatonin for at least seven 24 h light/dark cycles under these conditions. In this complementary study, the morphofunctional state of the photoreceptor cells was examined 1) by electron (transmission, scanning) microscopy, and 2) by pharmacological tests under different lighting conditions. Using polycarbonate membranes together with RPMI 1640 medium appeared the most suitable. The segmented organization of photoreceptor cells was well preserved when using the culture-treated membranes. It tended to disappear in cells embedded in the collagen gel and was lost after passage through the Percoll gradient. However, this one allowed obtention of an homogeneous population of photoreceptors, as recognized by their intracellular components. Intracellular organelles were rather well preserved in the embedded photoreceptors. The study also provides novel information on the nature of second messengers involved in the photoperiodic control of melatonin production in photoreceptor cells. From the effects of an adenylyl cyclase activator and a phosphodiesterase inhibitor it appeared that 1) total cells and Percoll-selected cells behaved similarly, 2) the nocturnal rise in melatonin secretion was associated with an increase in cAMP content, and 3) a fall in cAMP may be a mechanism through which light reduces melatonin secretion by photoreceptor cells. Cyclic GMP, the metabolism of which also appeared to be controlled by light, did not seem involved in the photoperiodic control of melatonin production. The method proposed herein offers interesting perspectives for the study of the photoneuroendocrine properties of isolated photoreceptor cells.

Trout pineal cells were dissociated using a trypsin-DNase digestion technique. An enriched population of photoreceptor cells was selected from a Percoll gradient centrifugation. The ability of cultured photoreceptor cells (selected or not on a Percoll gradient) to produce melatonin rhythmically was investigated during seven 24 h light/dark cycles. During each cycle, trout pineal photoreceptor cells released low amounts of melatonin during daytime and high amounts during night-time. Under continuous darkness, melatonin release was continually high. The profile of its rhythm and that of the activity of the hydroxyindole-O-methyltransferase-the last enzyme of the melatonin biosynthetic pathway-depended on the substrates and on the culture media used. Some of them appear suitable for short- or long-term culture of photoreceptor cells permitting the study of their neuroendocrine properties.

1. Chickens kept in constant light, as opposed to constant darkness, display a twofold increase in the activity of pineal hydroxyindole-O-methyltransferase (HIOMT), the last acting enzyme in the melatonin pathway. 2. Using an immunological approach, we presently show that this regulation of HIOMT activity reflects changes in the concentration of a single molecular form of the enzyme protein (a 38 kDa polypeptide). Immunohistofluorescence indicates that these concentration changes concurrently affect modified photoreceptors and pinealocyte-like cells in the chicken pineal organ. 3. Together, the present data support the hypothesis that environmental lighting might regulate the expression of the HIOMT gene.

Hydroxyindole O-methyltransferase (EC 2.1.1.4) is the enzyme that catalyses the synthesis of melatonin in the pineal gland and in the retina. Polyadenylated RNA from chicken pineal glands was used to prepare a cDNA library in lambda gt11. The library was screened with an antiserum directed against chicken hydroxyindole O-methyltransferase, and one cDNA clone was isolated. The fusion protein expressed by phage lysogens was identified on Western blots as a 165 kDA immunoreactive protein (beta-galactosidase, 110 kDa; hydroxyindole O-methyltransferase, 38 kDa). The fusion protein exhibited hydroxyindole O-methyltransferase activity. Its Km values for N-acetyl-5-hydroxytryptamine and S-adenosylmethionine were 5 times those of the natural enzyme. The intrinsic activity of the fusion protein was approx. 0.25% that of the natural enzyme. The cDNA consisted of 1436 nucleotides, including a 1038-nucleotide sequence encoding a full-length 346-amino-acid hydroxyindole O-methyltransferase. Comparison with bovine hydroxyindole O-methyltransferase [Ishida, Obinata & Deguchi (1987) J. Biol. Chem. 262, 2895-2899] revealed 52% identity in nucleotide sequences and 44% identity in peptide sequences. Northern-blot analysis revealed the presence of hydroxyindole O-methyltransferase mRNA transcripts in chicken pineal gland and retina, but not in the telencephalon.

Abstract A suspension culture of ovine pineal cells was developed to investigate the regulation of melatonin synthesis and release. Dosedependent stimulation of melatonin release by a series of adrenergic agonists yielded a typical beta-adrenergic profile. Serotonin N-acetyltransferase (NAT), the rate-limiting enzyme in melatonin biosynthesis, was also stimulated by a beta-adrenergic mechanism. However, NAT activity appeared less sensitive than melatonin release to beta-adrenergic stimulation. No evidence was obtained for a contribution of alpha(1)-adrenergic receptors to the regulation of NAT activity and melatonin release. Activation of adenylate cyclase by forskolin or addition of a cyclic AMP analogue increased both melatonin release and NAT activity. In contrast, the Ca(2+) ionophore A(23187) stimulated melatonin release without a detectable increase in NAT activity. Together, the present data argue for a beta-adrenergic regulation of both NAT activity and melatonin release in ovine pinealocytes. The evidence also suggests that two intracellular mechanisms may control melatonin release in ovine pinealocytes: a cyclic AMP-dependent mechanism, associated with an increase in NAT activity and a Ca(2+)-dependent mechanism, independent of NAT activity.

The pineal organ of fish contains photoreceptor cells with structural and functional analogies to retinal photoreceptors. In these cells, the light/dark (LD) cycle influences the production of melatonin by controlling the activity of one of its synthetizing enzymes, serotonin N-acetyltransferase (NAT). The daily rhythm in NAT activity is generated endogenously in the pike but not in the trout pineal. We report here that in addition to the LD information, chemical factors are also involved in the control of melatonin production. Adenosine and two of its analogs stimulated or inhibited NAT activity and melatonin release in cultured pike and trout pineals, depending on the experimental conditions. It is believed that the nucleoside, produced locally, exerts a modulatory role on the neurohormonal output via still enigmatic mechanisms, involving a transmembranous carrier. Nocturnal melatonin production in cultured pike pineals was inhibited by alpha-adrenergic agonists and stimulated by a beta-adrenergic agonist. No effect could be induced in trout pineals cultured under similar conditions. Because melatonin production by pineal photoreceptors is apparently regulated by both light and chemical inputs, we propose they might be multieffector cells.

The pineal gland influences circadian activity and seasonal breeding through the production of an indolic hormone, melatonin. The terminal step of melatonin biosynthesis is catalyzed by hydroxyindole-O-methyltransferase (HIOMT). Using an antibody directed against HIOMT, we examined the differentiation of the melatoninergic phenotype in the developing chick pineal gland. HIOMT first appeared 4 days before hatch and rose linearly until the 7th day posthatch. This was correlated with an increased immunoreactivity of the 38 kDa enzyme on Western blots and with an accelerated rate of HIOMT biosynthesis as demonstrated by [35S]methionine labeling. Immunocytochemistry revealed a growing number of HIOMT-positive cells between day 2 before hatch and day 15 posthatch. Until hatching HIOMT was expressed almost exclusively in modified photoreceptors. Parafollicular pinealocytes became HIOMT-positive mostly after hatching. Their different timings of functional differentiation emphasize the existence of two populations of melatonin-producing cells in the chick pineal gland.

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.

Eighteen homodigital island flaps were performed for pulp defects over a period of two years in the Hand Emergency unit. Apart from one failure due to an incorrect indication, the results confirmed the excellent trophicity of the flap and its aesthetic appearance was appreciated by the 17 patients. Discrimination on Weber's test, the possibilities of advancement, and the fact that the size of the flap can be tailored to individual needs make it one of the most attractive alternatives in this type of indication. However, postoperative stiffness of the PIP joint is an almost constant disadvantage requiring an extension splint as soon as possible. The homodigital island flap is an elegant, minimally disfiguring flap indicated in pulp defects or distal palmar or lateral pulp amputations.

We investigated the role of cyclic AMP (cAMP) in alpha 2- and possible beta-adrenergic regulation of arylalkylamine-N-acetyltransferase (NAT), the penultimate enzyme in the biosynthesis of melatonin. The study was performed on primary cultures of dispersed chick pineal cells. Electron microscopy indicated that approximately 70% of the dispersed cells were modified photoreceptors. A similar proportion of melatoninergic cells was detected by immunocytochemical labeling of hydroxyindole-O-methyltransferase, the final enzyme in the biosynthesis of melatonin. Adrenergic agonists caused a sustained 50% inhibition of forskolin-augmented cAMP levels and NAT activity, with an alpha 2-adrenergic potency order of UK 14,304 greater than or equal to clonidine greater than norepinephrine greater than phenylephrine. Noradrenergic inhibition of 3-isobutyl-1-methylxanthine-augmented cAMP levels and NAT activity was reversed by yohimbine (an alpha 2-adrenergic antagonist) but not by prazosin (an alpha 1-adrenergic antagonist). The alpha-adrenergic inhibition of cAMP accumulation and NAT activity was prevented by pertussis toxin. Addition of propranolol (a beta-adrenergic antagonist) was necessary to observe an inhibitory effect of norepinephrine on cAMP levels but not on NAT activity. Similarly, the beta-adrenergic agonist isoproterenol transiently increased cAMP levels but did not affect NAT activity. The data indicate that the alpha 2-adrenergic inhibition of NAT activity in chick pineal cells is strongly correlated with an inhibition of cAMP accumulation. The lack of beta-adrenergic effect on NAT suggests that beta-adrenoceptors might be on a subset of cells that do not produce melatonin or that the beta-adrenergic-induced increase in cAMP levels is too transient to affect NAT.

The modified photoreceptor cell (MP), homologous to the cone-like photoreceptor, is the principal cell type of the avian pineal organ. Two types of secretions (indoles and specific proteins-- pinealins ) are produced in this polarized cell. The synthesis of indoles undergoes striking circadian and circannual changes. MP appear to be receptoneuroendocrine cells which transduce circadian information into 5- methoxyindolic (melatonin, 5-methoxytryptophol, 5-methoxytryptamine) and possibly peptidic neurohormones. Taking into account different stages of differentiation and/or different physiological states (due to cyclic changes) for MP, several mechanisms of transduction of photoperiodic inputs into circadian hormonal outputs have been hypothesized.

Histochemical fluorescence (Falck-Hillarp) and microspectrofluorometric (MSF) methods were used to characterize different types of catecholamine-containing cellular elements located in the reproductive system of fresh-water snails (Bulinus truncatus, Planorbarius corneus) and land snails [Archachatina marginata, Helix (Cryptomphalus) aspersa]. Transverse sections through the genital tract display a common structural pattern of tubular differentiations: (1) an internal epithelium bordering the lumen and containing variable numbers of monoaminergic cells; (2) an enveloping sheath of connective and muscular tissue containing fine nerve fibers in the form of a network that exhibits a variable degree of density. MSF determinations showed that the formaldehyde-induced fluorophores of the intraepithelial aminergic cells belong to the following classes: (1) the DOPA/dopamine group in the duct of the albumen gland of B. truncatus and the carrefour of A. marginata; and (2) the norepinephrine/epinephrine group in the duct of the albumen gland and in the oviduct sac of P. corneus. In the reproductive systems of B. truncatus and P. corneus (duct of the albumen gland, oviduct sac, vagina), A. marginata and H. aspersa (duct of the fertilization pocket, origin of the receptaculum seminis, carrefour), the MSF analysis revealed norepinephrine/epinephrine-containing intramural nerve fibers. On the other hand, the small neurons in the vagina of B. truncatus belong to the DOPA/dopamine group.

Using two immunocytochemical procedures (i.e., immunofluorescence and the unlabeled peroxidase-antiperoxidase method), the localization of a serotonin(HT)-like and of a N-acetylserotonin (aHT)-like immunoreactivity in the pineal organ of the pike was studied during winter. It was shown that immunostaining was exclusively restricted to the cells of the receptor line (CRL = typical and modified photoreceptors). The intensity of the reactions varied through the light-dark cycle, HT-like immunoreactivity being high during the photophase and low during the scotophase. In contrast, aHT-like immunoreactivity was highest at the beginning of the scotophase. HT and aHT-like immunoreactivities were detected in all cell types of the pineal epithelium after administration of a monoamine oxidase inhibitor. Up to now, only HT immunoreactivity could be localized at the ultrastructural level. In a number of typical and modified photoreceptors, a HT-positive staining seemed to be confined within the hyaloplasm of the inner segment, particularly with that of the perikaryon and basal pedicle. Our previous and present results strongly suggest that indole compounds, which are involved in the regulation of various neuroendocrine processes in fish, are synthetized within the CRL. Taking into account that the CRL of the pike are also photosensitive, it appears more and more likely that they are photoneuroendocrine cells involved in mediating the effects of the photoperiod on various physiological and behavioral processes.

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.

Circadian variations in melatonin content have been studied in the pineal organ, retina and plasma of adult pigeons using radioimmunoassay. A clear-cut peak of melatonin was observed in the pineal organ at middark; this middark peak was of larger amplitude in July than in November. A nocturnal rise in melatonin concentration was also observed in the retina and plasma. We also used an immunocytochemical technique revealing the positive reactions of a melatonin-like compound in the photoreceptor cell layer (inner segments) of the pigeon's retina. Up to now, no melatonin storage has been visualized in the pineal organ of the pigeon.

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 the amount of [3H]-5-hydroxyindoleacetic acid decreased, that of [3H]-5-hydroxytryptophol decreased faster than that of [3H]-5-methoxytryptophol, and 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. At the beginning of the photophase or at the onset of darkness, the uptake and metabolism of [3H]melatonin (after a 10-min pulse followed by a 10-min incubation in cold medium) resulted mainly in the formation of [3H]-5-methoxytryptophol (23 to 43%) and of [3H]-5-methoxytryptamine (6 to 12%). These results show that the pike pineal organ can synthesize all indoles that are known in the pineal gland of higher vertebrates. Usual, but also unusual, pathways of the indole metabolism were found that will need further clarification. Among these are the possible carboxylation of serotonin and deacetylation of melatonin (leading to the synthesis of 5-methoxytryptophol). Altogether, the results obtained suggest that the indole metabolism might be more complex than what has already been described in vertebrates.

Since high-resolution radioautography (dipping technique) might be very useful for the study of indole metabolism in the pineal cells, the retention of [3H]-indoles has to be examined during the preparation of specimens for electron microscopy (EM). The pineal organ of the parakeet (Melopsittacus undulatus) was used in the present work. 1) Indole metabolism: following uptake of [3H]-5-hydroxytryptophan ([3H]-HW) in vivo and [3H]-5-hydroxytryptamine ([3H]-HT) in vitro in similar seasonal and nycthemeral conditions--all the known pineal indolic metabolites were recovered by thin layer chromatography. [3H]-5-methoxyindoles were also formed from [3H]-melatonin ([3H]-aMT). 2) The radioactivity of fluids used in the processing of pineal organs in EM was determined by liquid scintillation counting: (a) no exogenous [3H]-indoles could be revealed in EM solutions after [3H]-HW in vivo uptake. (b) 8.8 to 13.4% of [3H]-indoles were washed out by glutaraldehyde after [3H]-HT in vitro uptake. (c) most of the 5-methoxyindoles after in vitro uptake of [3H]-aMT were lost in glutaraldehyde. Our chromatography procedures did not permit the identification of [3H]-indoles extracted by the glutaraldehyde fixative. In previous experiments, [3H]-HW and [3H]-HT uptake showed the presence of selective radioautographic reactions in the cells of the receptor line; however, silver grains were scarce and diffusely distributed in the pineal parenchyma after [3H]-aMT uptake.

In the pigeon pineal organ studied in vitro, high resolution radioautography was used to visualize the sites of uptake and retention of [3H]-indoles: --incubation of the pineal organ with [3H]-5-hydroxytryptophan ([3H]-5-HTP) during the day and with [3H]-5-hydroxytryptamine ([3H]-5-HT) during the night led to strong selective labelling of the receptor line cells (RLC) which correspond to modified photoreceptor cells (rudimentary photoreceptor cells). No significant radioautographic reactions were found in other cell types. When the 20 min radioactive signal was followed by 20-100 min post-incubation in a cold medium, labelling was still restricted to RLC. The in vitro study of indole metabolism sites might be facilitated by this preparation. --labelling after [3H]-melatonin incorporation was found mainly in RLC but also in interstitial cells. Reactions were less intense than those obtained with [3H]-hydroxyindoles although some erythrocytes were strongly labelled. No definite conclusion can be drawn from the radioautographs, in which artefacts were apparent.

Biosynthesis of the indolic hormone melatonin has been reported in the pineal gland and retina. The terminal step of melatonin synthesis is catalysed by hydroxyindole-O-methyltransferase (HIOMT), an enzyme displaying highest levels of activity in the pineal gland and retina. Several laboratories have suggested that melatonin synthesis might take place in retinal photoreceptors and in photoreceptor-derived cells of the pineal gland. Experimental support to this hypothesis is progressively building up with the immunocytochemical identification of HIOMT-containing cells in various animal species. In the present report, HIOMT was purified from the chicken pineal gland using a one-step chromatographic procedure and an antiserum against the enzyme was obtained in the rabbit. The antiserum was further purified by immunoadsorption chromatography on chicken brain proteins. Using electroblots immunochemical labeling, HIOMT from chicken pineal gland and retina was identified as a 38-kDa protein. Pineal HIOMT was further resolved into components of different pHi-values (5.4-5.7 and 6.8), using two-dimensional gel electrophoresis. Immunoprecipitation of HIOMT activity was observed in pineal homogenates and, for the first time, in homogenates of the retina. Immunofluorescence microscopy provided the first evidence that HIOMT is contained in modified photoreceptors of the chicken pineal gland. No immunofluorescence could be observed in the retina, maybe due to the lower level of HIOMT activity in this tissue. Together, the data indicate that the antiserum may be a useful tool to study the regulation of HIOMT synthesis in the pineal gland and in the retina. Further work is required to identify HIOMT-containing cells in the retina.

The present investigation sought to characterize the adrenergic inhibition of arylalkylamine-N-acetyltransferase in cultured chicken pineal glands. Arylalkylamine-N-acetyltransferase, the melatonin rhythm generating enzyme, displays daily oscillations of activity that are driven by a circadian oscillator. Norepinephrine released at sympathetic nerve endings inhibits the enzyme and appears to play a role in maintaining a circadian rhythm of melatonin release. Chicken pineal glands were isolated in organ culture and the effects of adrenergic agents on the night time peak of N-acetyltransferase activity were studied. Norepinephrine and clonidine prevented 50 to 65% of the nocturnal rise of N-acetyltransferase activity. When applied at middark, norepinephrine and clonidine caused a 50 to 65% inhibition of N-acetyltransferase activity in 2 hours. Dose-response studies indicated clonidine was 100 times more potent than norepinephrine or cirazoline at inhibiting N-acetyltransferase activity. Inhibition of N-acetyltransferase activity was also observed, at micromolar concentration with epinephrine, UK 14,304 and alpha-methylnorepinephrine but not with phenylephrine, isoproterenol or dopamine. Epinephrine and clonidine actions were antagonized by yohimbine but not by prazosin. Destruction of the presynaptic compartment by bilateral superior cervical ganglionectomy did not affect the clonidine-induced inhibition of N-acetyltransferase and its reversal by yohimbine. It is concluded that the adrenergic inhibition of N-acetyltransferase activity in chicken pineal gland probably occurs via stimulation of postsynaptic alpha 2-adrenergic receptors.

The effects of adenosine, of its non-metabolizable analogs, and of compounds related to its metabolism, were investigated in the photosensitive chicken pineal, maintained in static culture or in superfusion. Stimulation or inhibition of melatonin production was obtained, depending on the experimental conditions tested. Endogenous adenosine is involved in the regulation of the melatonin output. The effects of the nucleoside might depend on the balance between its intra- and extracellular pools; (re)uptake mechanisms are most probably involved. It is suggested that cell surface receptors mediate adenosine effects, but intracellular actions (P-site, transmethylation pathways) are not excluded. This investigation is a breakthrough in the field of pineal physiology which offers new perspectives in the study of the control of melatonin production.

The authors report their first evaluation of 40 arthroplasties of the wrist with the Swanson implant. The indication for this procedure was principally severe damage to the wrist joint. The radiologic findings have been summarized. In regard to operative technic, there have been no major problems. In 20 cases also arthroplasty of the head of the ulnar was done. There were no significant early complications. The clinical results after 14 months, with a range of 6 to 45 months, was always satisfactory in regard to pain and the preservation useful flexion and extension. So far, tolerance to the implant seems to be satisfactory. The joint narrowing, regularily found in the new articulation, does not detract from the long-term results. Thus the authors retain a favorable impression of this arthroplasty which should find a place in the treatment of badly damaged rheumatoid wrists. In addition to relieving the pain, it preserves mobility that would be sacrificed with an arthrodesis. Among the associated procedures, the authors have been disappointed with arthroplasty of the head of the ulna and have given it up.

Photoperiodic control of several biological rhythms is exerted through the inhibitory effect of light on melatonin synthesis in the pineal organ. Hydroxyindole-O-methyltransferase (HIO-MT), the last acting-enzyme in melatonin biosynthesis, constitutes a specific marker of melatoninergic cells. In the present study, an antibody directed against chicken HIOMT was affinity-purified and used to identify melatoninergic cells in the pineal organ of chicken, quail, sparrow and blackbird. Regardless of the species, intense immunocytochemical reactions were observed in modified photoreceptors, whereas other cellular constituents (mostly glial cells) remained unlabeled. We conclude that modified photoreceptors synthesize melatonin in the avian pineal gland and are thus accountable for the translation of the photoperiodic input into hormonal output.

In the pineal gland of the mouse the distribution of serotonin (5-HT) and its eventual relationships to a protein secretion were examined by means of fluorescence histochemical (Falck-Hillarp) and ultracytochemical (chromaffin and argentaffin) methods. (1) Yellow formaldehyde-induced fluorophores, characteristic of high concentrations of 5-HT, were found in pinealocytes, interstitial cells and sympathetic adrenergic nerve endings. The 5-HT content was studied according to the circadian variations and different drug treatments. (2) By use of ultracytochemical methods in untreated control mice precipitates indicative of 5-HT were found over dense-core vesicles (DCV; mean diameter: 100 nm) of the pinealocytes and the vesicular compartment of the sympathetic nerve fibers. After reserpine and p-CPA treatments, the reactivity of the DCV disappeared, but the protein secretion accumulated within the DCV was still present. After nialamide treatment the precipitates in the DCV increased and, similar to control mice, masked the protein secretion. Taking into account the specificity and sensitivity of these three complementary methods, previous biochemical data in mammals and ultracytochemical data in submammalian vertebrates, synthesis, storage, catabolism and release of 5-HT in the mouse and hamster pineals are discussed at the cellular level. Different pools of 5-HT are present: agranular and granular in pinealocytes and sympathetic nerve endings; agranular in interstitial cells. In the mouse, only a small portion of the total 5-HT content appears to bae secondarily taken up by the DCV. Within the DCV, 5-HT is possibly bound to a protein secretion of unknown significance (peptidergic neurohormone?). In the pinealocytes, which are sensitive to a large variety of inputs, processes of indole and protein secretion are found. These cells apparently are the recepto-secretory elements of the mammalian pineal gland. They can thus be classified as a member of the diffuse neuroendocrine system of the paraneuron group.