Showing papers on "Pinealocyte published in 1997"

The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland.

Abstract: A remarkably constant feature of vertebrate physiology is a daily rhythm of melatonin in the circulation, which serves as the hormonal signal of the daily light/dark cycle: melatonin levels are always elevated at night. The biochemical basis of this hormonal rhythm is one of the enzymes involved in melatonin synthesis in the pineal gland-the melatonin rhythm-generating enzyme-serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, E.C. 2.3.1.87). In all vertebrates, enzyme activity is high at night. This reflects the influences of internal circadian clocks and of light. The dynamics of this enzyme are remarkable. The magnitude of the nocturnal increase in enzyme activity ranges from 7- to 150-fold on a species-to-species basis among vertebrates. In all cases the nocturnal levels of AA-NAT activity decrease very rapidly following exposure to light. A major advance in the study of the molecular basis of these changes was the cloning of cDNA encoding the enzyme. This has resulted in rapid progress in our understanding of the biology and structure of AA-NAT and how it is regulated. Several constant features of this enzyme have become apparent, including structural features, tissue distribution, and a close association of enzyme activity and protein. However, some remarkable differences among species in the molecular mechanisms involved in regulating the enzyme have been discovered. In sheep, AA-NAT mRNA levels show relatively little change over a 24-hour period and changes in AA-NAT activity are primarily regulated at the protein level. In the rat, AA-NAT is also regulated at a protein level; however, in addition, AA-NAT mRNA levels exhibit a 150-fold rhythm, which reflects cyclic AMP-dependent regulation of expression of the AA-NAT gene. In the chicken, cyclic AMP acts primarily at the protein level and a rhythm in AA-NAT mRNA is driven by a noncyclic AMP-dependent mechanism linked to the clock within the pineal gland. Finally, in the trout, AA-NAT mRNA levels show little change and activity is regulated by light acting directly on the pineal gland. The variety of mechanisms that have evolved among vertebrates to achieve the same goal-a rhythm in melatonin-underlines the important role melatonin plays as the hormonal signal of environmental lighting in vertebrates.

Melatonin receptors : molecular biology of a new family of g protein-coupled receptors

Abstract: A family of high-affinity, G protein-coupled receptors for the pineal hormone melatonin has been cloned from vertebrates. These recombinant receptors exhibit similar affinity and pharmacological characteristics to each other and to endogenous receptors, as defined with the melatonin agonist 2-[ 125I]iodomelatonin (125I-Mel). Two mammalian melatonin receptor subtypes have been identified by molecular cloning studies. The mammalian Mel1a melatonin receptor is expressed in most sites containing 125I-Mel binding. This includes the hypothalamic suprachiasmatic nucleus and hypophyseal pars tuberalis, presumed sites of the circadian and some of the reproductive actions of melatonin, respectively. The mammalian Mel 1b melatonin receptor is expressed in retina and brain and may mediate the reported effects of melatonin on retinal physiology in some mammals. A third receptor subtype, the Mel1c melatonin receptor, has been cloned from zebra fish, Xenopus, and chickens but not from mammals. Molecular cloning of a mel...

The pineal organ of teleost fishes

Abstract: The pineal organ of teleost fish is a directly photosensory organ that contains photoreceptor cells similar to those of the retina. It conveys photoperiod information to the brain via neural pathways and by release of indoleamines, primarily melatonin, into the circulation. The photoreceptor cells respond to changes in ambient illumination with a gradual modulation of neurotransmission to second-order neurons that innervate various brain centres, and by modulation of indoleamine synthesis. Melatonin is produced rhythmically, and melatonin synthesis may be regulated either directly by ambient photoperiod, or by an endogenous circadian oscillator that is entrained by the photoperiod. During natural conditions, melatonin is produced at highest levels during the night. Although the pineal organ undoubtedly influences a variety of physiological parameters, as assessed by experimental removal of the pineal organ and/or administration of exogenous indoleamines, its role in any physiological situation is not clear cut. The effects of any interference with pineal functions appear to vary with the time of year and experimental photothermal regimes. There are strong indications that the pineal organ is one component in a central neural system that constitutes the photoperiod-responding system of the animal, i.e. the system that is responsible for correct timing of daily and seasonal physiological rhythms. It is important to envisage the pineal organ as a part of this system; it interacts with other photosensory structures (the retina, possibly extraretinal non-pineal photoreceptors) and circadian rhythm generators

Developmental Expression Pattern of Phototransduction Components in Mammalian Pineal Implies a Light-Sensing Function

Abstract: Whereas the pineal organs of lower vertebrates have been shown to be photosensitive, photic regulation of pineal function in adult mammals is thought be mediated entirely by retinal photoreceptors. Extraretinal regulation of pineal function has been reported in neonatal rodents, although both the site and molecular basis of extraretinal photoreception have remained obscure. In this study we examine the developmental expression pattern of all of the principal components of retinal phototransduction in rat pineal via cRNA in situ hybridization. All of the components needed to reconstitute a functional phototransduction pathway are expressed in the majority of neonatal pinealocytes, although the expression levels of many of these genes decline dramatically during development. These findings strongly support the theory that the neonatal rat pineal itself is photosensitive. In addition, we observe in neonatal pinealocytes the expression of both rod-specific and cone-specific phototransduction components, implying the existence of functionally different subtypes of pinealocytes that express varying combinations of phototransduction enzymes.

Chick pineal clock regulates serotonin N-acetyltransferase mRNA rhythm in culture

Abstract: Melatonin production in the chick pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT; EC 2.3.1.87), the penultimate enzyme in melatonin synthesis. In contrast to the external regulation of pineal rhythms in mammals by the suprachiasmatic nucleus, rhythmic changes in AA-NAT activity in cultured chick pineal cells are controlled by an oscillator located in the pineal cells themselves. Here we present evidence that the chick pineal clock generates a rhythm in the abundance of AA-NAT mRNA in cultured cells that parallels the rhythm in AA-NAT activity. In contrast, elevating cAMP by forskolin treatment markedly increases AA-NAT activity without producing strong changes in AA-NAT mRNA levels, and lowering cAMP by norepinephrine treatment decreases enzyme activity without markedly decreasing mRNA. These results suggest that clock-controlled changes in AA-NAT activity occur primarily through changes at the mRNA level, whereas cAMP-controlled changes occur primarily through changes at the protein level. Related studies indicate that the clock-dependent nocturnal increase in AA-NAT mRNA requires gene expression but not de novo protein synthesis, and that AA-NAT mRNA levels are suppressed at all times of the day by a rapidly turning over protein. Further analysis of the regulation of chick pineal AA-NAT mRNA is likely to enhance our understanding of the molecular basis of vertebrate circadian rhythms.

Time and Time Again: The Phylogeny of Melatonin as a Transducer of Biological Time

Abstract: The circadian secretion of melatonin is a critical component in circadian and seasonal rhythms in many vertebrate species. This hormone is produced by photoreceptors and cell types derived from photoreceptors in vertebrate retinas and pineal complexes via circadian regulation of the biosynthetic enzymes arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase at both transcriptional and posttranscriptional levels. The question of whether other multicellular animals and organisms from other taxa produce melatonin in a homologously regulated pathway is at this point unclear, but preliminary evidence suggests that vertebrate and insect melatonin are produced by convergent or parallel phylogenies. The existence and function of algal and plant melatonin is worthy of further study but is unresolved at this point. In vertebrates, the role of melatonin in behavioral and systems physiology follows two phylogenetic patterns. First, the circadian regulation of visual system structures, including the ...

Stimulation of a nicotinic ACh receptor causes depolarization and activation of L-type Ca2+ channels in rat pinealocytes.

Abstract: 1 Membrane voltage (Vm) recordings were obtained from isolated rat pinealocytes using the patch-clamp technique In parallel to the electrophysiological experiments, intracellular Ca2+ measurements were performed using fura-2 2 The resting Vm averaged -43 mV and replacement of extracellular NaCl by KCl completely depolarized the cells This indicates that the resting Vm is dominated by a K+ conductance Single-channel recordings revealed the presence of a large conductance Ca(2+)-activated charybdotoxin-sensitive K+ channel 3 Application of ACh (100 microM) depolarized the pinealocytes on average by 16 mV The depolarizing effect of ACh was mimicked by nicotine (50 microM) and was prevented by tubocurarine (100 microM) 4 The ACh-induced depolarization was largely abolished in the absence of extracellular Na+, but was not significantly affected by extracellular Ca2+ removal 5 Application of ACh (100 microM) caused an increase in [Ca2+]i This increase was completely dependent on the presence of extracellular Ca2+ and was largely reduced after extracellular Na+ removal Nifedipine (1 microM) reduced the ACh-induced increase in [Ca2+]i by about 50% 6 Our findings indicate that in rat pinealocytes stimulation of a nicotinic ACh receptor (nAChR) induces depolarization mainly by Na+ influx via the nAChR The depolarization then activates L-type Ca2+ channels, which are responsible for the nifedipine-sensitive portion of the intracellular Ca2+ increase Ca2+ influx via the nAChR probably also contributes to the observed rise in [Ca2+]i

Localization and diurnal expression of mRNA encoding the β1-adrenoceptor in the rat pineal gland: an in situ hybridization study

Abstract: The rat pinealocyte is stimulated by norepinephrine, which is released from sympathetic nerve fibers innervating the gland. Norepinephrine binds to β1-adrenoceptors situated on the pinealocyte cell membrane. Ligand binding to these receptors exhibits a diurnal rhythm, with the largest number occurring in the late part of the light phase when the release of norepinephrine is minimal. By using in situ hybridization with a cDNA antisense oligonucleotide probe recognizing mRNA encoding the rat β1-adrenoceptor, we have demonstrated a stronger hybridization signal in the rat pineal gland than in other brain regions. Cells containing β1-mRNA are located in the superficial pineal gland, the deep pineal gland, and the pineal stalk. However, the number of receptors varies considerably between the individual pinealocytes. The β1-mRNA in situ hybridization signal for mRNA encoding the β1-adrenoceptor of the rat pineal has been quantified over a 24-h period; the strongest signal is found at mid-dark and the weakest signal at mid-light, indicating that the transcriptional regulation of β1-mRNA synthesis in the rat pineal is diurnal. In addition, maximal receptor protein expression occurs late in the light phase in the rat pineal and is thus considerably delayed compared with its peak mRNA synthesis. This lag time indicates that the β1-receptor is regulated at the translational or post-translational level. Removal of the sympathetic input to the pineal gland by superior cervical ganglionectomy decreases the β1-mRNA signal in the gland.

Molecular cloning and localization of rhodopsin kinase in the mammalian pineal

Abstract: Several retinal photoreceptor proteins involved in phototransduction have also been found in the mammalian pineal. This study demonstrates that rat and human pineals express protein kinases that are identical to the corresponding rod photoreceptor rhodopsin kinases. The deduced amino acid sequence of rat and human rhodopsin kinases have 84% sequence similarity to the earlier reported sequence of the bovine retinal enzyme, with complete conservation of the topological regions containing the position of the catalytic domain and sites of posttranslational modifications. Rat pineal also expresses rod opsin and putative blue cone opsin. Using immunocytochemistry, rod opsin and rhodopsin kinase were found to be co-localized in pinealocytes in the human tissue. These data demonstrate that the mammalian pineal contains light-sensitive opsins and a kinase involved in their inactivation. These findings correlate with an earlier report that neonatal rats show extraretinal light sensitivity, and suggest that a functional photoreceptive system may be present in the adult mammalian pineal.

The role of the intracellular and extracellular serotonin in the regulation of melatonin production in rat pinealocytes.

Abstract: This study investigated whether the activation of pinealocyte beta-adrenergic receptors is involved in the regulation of serotonin (5-HT) synthesis and release, as it is for melatonin production. In addition, the role of the intra- and extra-cellular 5-HT in modulating the synthesis of melatonin induced by the beta-adrenergic agonist isoproterenol (ISO) was also studied. The incubation of dissociated pinealocytes with 0.1-10 microM ISO resulted in a concentration-dependent increase of melatonin synthesis. 5-HT release and intracellular 5-HT content were increased by 0.1 and 1 microM ISO but they were reduced after ISO 10 microM. Moreover, when incubated with the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA), the secretion of 5-HT as well as the intracellular 5-HT levels were markedly reduced in both ISO-stimulated and unstimulated conditions. Melatonin release was also inhibited by PCPA, although it responded in the expected manner to increasing concentrations of ISO. These data indicate that the release of 5-HT from pinealocytes depends on the availability of cytoplasmic 5-HT, which in turn is highly dependent on the tryptophan hydroxylase activity. In cells stimulated with moderate ISO concentrations, 5-HT release may be an important regulatory process of pineal 5-HT. After a large stimulation of N-acetyltransferase (NAT) activity by ISO, the synthesis of melatonin prevails on 5-HT release, whose decrease is associated to a deficit of intracellular 5-HT. On the other hand, the present study shows that the incubation of pineal cells with high concentrations of 5-HT or with a selective 5-HT2 receptor agonist, alpha-methyl-5-hydroxytryptamine, reverses partially the inhibitory effect of PCPA on the ISO-stimulated melatonin synthesis. In contrast the 5-HT2 antagonist, ketanserin, results in an inhibiton of the release of melatonin following ISO stimulation. These results suggest that released 5-HT may have a role in the full expression of the beta-adrenergically induced NAT activity and, thus, may contribute to the optimal melatonin synthesis at night.

The pineal complex of reptiles: physiological and behavioral roles

Abstract: The pineal complex of reptiles is a morphologically and functionally connected set of organs that originates as an evagination of the roof of the diencephalon. It is formed by two structures: the pineal organ and the parietal eye. The pineal gland is present in Chelonia, Squamata and Rhynchocephalia, but not in Crocodilia. The parietal eye is present in some species of lizards (Squamata) and in the tuatara (Rhynchocephalia). Both the pineal gland and the parietal eye are photosensitive. In particular, the parietal eye is an highly organized photoreceptive structure, with a well-defined lens, cornea and retina. The most important (and studied) secretory product of this complex is the hormone melatonin which is synthesized by both organs (pineal and parietal eye). The pineal organ is believed to be the neuroendocrine transducer of changes in photoperiod and environmental temperature and it has been demonstrated to have a functional role in many aspects of reptilian biology. Melatonin has an influence on the...

Cellular and Molecular Regulation of Serotonin N-Acetyltransferase Activity in Chicken Retinal Photoreceptors

Abstract: Serotonin N-acetyltransferase (AA-NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and large changes in the activity of this enzyme appear to regulate the rhythm in melatonin synthesis. Recent advances have made it possible to study the mRNA encoding chicken AA-NAT, which has only been detected in the retina and pineal gland. Within the retina, AA-NAT mRNA is expressed primarily in photoreceptors. The levels of chicken retinal AA-NAT mRNA and activity exhibit 24-hour rhythms with peaks at night. These rhythms appear to reflect circadian clock control of AA-NAT mRNA abundance and independent effects of light and darkness on both mRNA levels and enzyme activity. The effects of darkness and light may occur through alterations in cAMP-dependent protein phosphorylation, which increases AA-NAT activity in photoreceptor cell cultures. The cAMP-dependent increase of AA-NAT enzyme activity reflects, at least in part, increased mRNA levels and inhibition of enzyme inactivation by a posttranslational mechanism. This review discusses a hypothetical model for the cellular and molecular regulation of AA-NAT activity by circadian oscillators and light in chicken retinal photoreceptor cells.

Melatonin Effects on Serotonin Synthesis and Metabolism in the Striatum, Nucleus Accumbens, and Dorsal and Median Raphe Nuclei of Rats

Abstract: This work examined the influence of the pineal gland and its hormone melatonin on the metabolism of serotonin (5-HT) in discrete areas of the forebrain, such as the Striatum and the nucleus accumbens, and the midbrain raphe. The content of 5-HT and its major oxidative metabolite, the 5-hydroxyindoleacetic acid (5-HIAA), as well as the in-vivo tryptophan hydroxylation rate were examined after long-term pinealectomy (one month) and daily melatonin treatment (500 μg/kg; twice daily for ten days) in pinealectomized rats. Pinealectomy did not alter 5-HT content in any of these brain areas, but it significantly increased the content of 5-HIAA in Striatum and the 5-HIAA/5-HT ratio in nucleus accumbens. The normal values of these parameters were recuperated after administration of exogenous melatonin, but it also increased the rate of tryptophan hydroxylation in both areas. In addition, melatonin treatment decreased the levels of 5-HIAA in dorsal raphe nucleus. These data suggest that the pineal gland, through the secretion of melatonin, modulates the local metabolism of 5-HT in forebrain areas by acting on the oxidative deamination. Moreover, melatonin injected in pinealectomized rats derives in a more extended effect than pinealectomy and induces a stimulation of 5-HT synthesis in the striatum, probably due to a pharmacological effect. These results point to the striatum as a target area for the interaction between pineal melatonin and the serotonergic function, and suggest a differential effect of the melatonin injected on areas containing serotonergic terminals and cell bodies, which may relevant for the mode of action of melatonin and its behavioral effects.

Immunohistochemical Localization of Gonadotropin and Gonadal Steroid Receptors in Human Pineal Glands

Abstract: Recently, we demonstrated that melatonin secretion was increased in male patients with GnRH deficiency and decreased to normal levels during testosterone treatment. These data suggested that gonadal steroids modulate melatonin secretion, probably by activating specific receptors in the pineal gland. We used immunohistochemistry to localize gonadotropin (LH and FSH) and gonadal steroid (androgens and estrogens) receptors in human pineal glands. Tissues were obtained at autopsy from 25 males, aged 19–87 yr, and five prepubertal children, aged 0.2–10 yr. Positive staining for all four types of receptors (LH, FSH, androgen, and estrogen) in the pineal parenchymal cells, pinealocytes, was evident in all 30 glands examined. Double staining revealed that nuclear receptors (androgen or estrogen) coexisted with cytoplasmatic receptors (LH or FSH) in the same cells. The results demonstrate the presence of gonadotropin and gonadal steroid receptors in human pinealocytes from infancy to old age.

Immunoelectron-microscopic investigation of the subcellular localization of pinopsin in the pineal organ of the chicken

Abstract: Pinopsin is a photoreceptive molecule cloned from the chicken pineal organ. An antibody highly specific for pinopsin was applied in light- and electron-microscopic immunocytochemical studies of the pineal organ of 1 to 2-month-old chickens. Intense immunoreactivity was found in the follicular lumen at the light-microscopic level. In addition, small immunoreactive spherical or fibrous structures were diffusely distributed at the parafollicular aspect of the pineal organ. To identify immunoreactive elements precisely, we used pre-embedding immunoelectron microscopy. These studies revealed immunoreactive outer segments of pinealocytes arranged closely side by side in the follicular lumina. The thin initial portion of the outer segment arose from a basal body located in the inner segment. Immunoreactive pear-shaped outer segments occupied small lumina. Follicular lumina displayed immunonegative arrays of whorl-like lamellar membranes. Occasionally, these immunonegative structures were surrounded by immunoreactive concentric lamellar complexes. In the parafollicular pineal parenchyma, long slender cilium-like structures or enlarged cilia and concentric lamellar arrays showed intense immunoreactivity. All immunoreactive structures observed in this study were considered to represent outer segments of pinealocytes of the chicken pineal organ.

Light, Neurotransmitters and the Suprachiasmatic Nucleus Control of Pineal Melatonin Production in the Rat

Abstract: There is considerable interest in the neuronal pathways involved in the generation and entrainment of circadian rhythms. We have monitored the output of the pineal gland via the urinary metabolite of

Expression of synaptic membrane proteins in gerbil pinealocytes in primary culture.

Abstract: Pinealocytes of various mammalian species contain abundant synaptic-like microvesicles (SLMVs) which are considered the endocrine equivalent of neuronal synaptic vesicles. Although the pinealocytes may thus be a suitable cellular model for experimental in vitro studies of SLMVs, nothing is known about the presence of SLMVs in isolated pinealocytes maintained under tissue culture conditions. In the present investigation, we prepared dissociated primary cultures of gerbil pinealocytes to study the expression and distribution of protein components of synaptic vesicles/SLMVs and the presynaptic plasmalemma in pinealocytes kept in vitro. Using immunofluorescence microscopy, we found that cultured pinealocytes readily expressed all synaptic membrane proteins investigated, i.e., synaptophysin, synaptotagmin I, synaptobrevin II, syntaxin I and SNAP-25. Punctuate immunoreactivity for the vesicle-associated proteins could be detected throughout the cell bodies of pinealocytes and was also distributed into all of their processes which began to develop within the first days in culture. Outgrowing processes exhibited growth cone-like structures which were enriched in synaptic vesicle-associated proteins. After 1 week in vitro, pinealocytes had frequently formed an elaborate network of long interwoven processes. Accumulations of synaptic vesicle-associated proteins were observed in varicosities and terminal swellings of the processes. The vesicle-rich process swellings often established synaptic-like process swellings often established synaptic-like contacts with somata and processes of other pinealocytes. Some of the pinealocyte processes possessed additional axon-like properties as demonstrated by their lack of immunoreactivity for the somato-dendritic marker MAP2 and the transferrin receptor. The comparison of the staining patterns for synaptophysin and the endocytotic marker transferrin receptor by confocal laser scanning microscopy revealed a largely differential intracellular distribution of the two proteins. This may indicate that a substantial fraction of pinealocyte SLMVs by-passes the early endosomal-related recycling pathway of SLMVs. Herewith, we have shown that isolated gerbil pinealocytes maintained in primary culture can acquire morphological and neurochemical traits which closely mimick those observed in vivo. In particular, these cultures permit experimental studies of the compartment of pinealocyte SLMVs which seem to make up a major secretory pathway for paracrine intrapineal communication.

Effects of sympathicolytic and sympathomimetic drugs on pineal ultrastructure in the domestic pig

Abstract: Lewczuk B, Przybylska-Gornowicz B. Effects of sympathicolytic and sympathicomimetic drugs on pineal ultrastructure in the domestic pig. J. Pineal Res. 1997; 23:198–208. © Munksgaard, Copenhagen Abstract The effects of a 4-day administration of two sympathicolytic (α-methyl-p-tyrosine, propranolol) and three sympathicomimetic (clorgyline, isoproterenol, dobutamine) drugs on the ultrastructure of the pineal gland in the domestic pig were examined. The morphological changes in the pinealocytes of the experimental groups mainly concerned cytoplasmic dense bodies, primary lysosomes, multivesicular bodies, dense core vesicles, and smooth endoplasmic reticulum. These structures seemed to be under direct or indirect control by the adrenergic nervous system. The relative volume of dense bodies in pinealocyte perikarya increased after administration of α-methyl-p-tyrosine as well as propranolol and decreased on treatment with clorgyline. The decrease in the relative volume of primary lysosomes in pinealocyte perikarya was noted in pigs treated with clorgyline and isoproterenol. The number of multivesicular bodies in pinealocyte perikarya decreased after administration of α-methyl-p-tyrosine. The numerical density of dense core vesicles in pinealocyte perikarya increased after treatment with clorgyline. Administration of isoproterenol and clorgyline resulted in the presence of wide cisterns of smooth endoplasmic reticulum. Narrow cisterns of this form of endoplasmic reticulum and lamellar whorls were observed after treatment with α-methyl-p-tyrosine in pinealocytes with large dense bodies. The presence of multilamellar structures was a characteristic feature of the pinealocytes of pigs treated with isoproterenol and dobutamine. Administration of α-methyl-p-tyrosine and clorgyline resulted in the appearance of pinealocytes which differed in number, size, and structure of dense bodies.