Pineal Cells Research Articles

Ciliary neurotrophic factor inhibits differentiation of photoreceptor-like cells in rat pineal glands in vitro

Ciliary neurotrophic factor (CNTF) is a unique member of the interleukin-6 (IL-6) family, whose receptor subunit for ligand binding is exclusively expressed in the nervous system and muscle. The role of CNTF in mammalian development remains unknown. We recently reported the specific expression of CNTF in the pineal gland and eyes. To further examine the expression pattern and role of CNTF in development, we prepared a polyclonal antibody against rat CNTF, performed western blotting with this antibody, and confirmed a strong and specific expression of the CNTF protein in pineal glands and a moderate expression in the eyes among the various tissues examined in newborn rats. In pineal organ cultures of newborn rats, exogenously added recombinant rat CNTF potently inhibited the differentiation of photoreceptor-like cells in a dose-dependent manner, while CNTF did not influence the survival of pineal cells. Among several cell growth factors known to have a similar effect in retinal cultures examined, strong inhibitory effects were seen only with CNTF and the leukemia inhibitory factor (LIF), both of which belong to the IL-6 cytokine family. This inhibitory effect was the strongest during three to 6 days of culture when CNTF was added to these cultures. These results suggest that CNTF plays an inhibitory role in the development of photoreceptor-like cells in early postnatal rat pineal glands.

Purification and immunohistochemical analysis of calcium-binding proteins expressed in the chick pineal gland.

The pineal gland is a site of melatonin production, of which intracellular calcium ions (Ca2+) are likely involved in various aspects. To investigate the identity of molecules responsible for the Ca2+-dependent processes in the pineal cells, we prepared a cellular extract from 2000 chick pineal glands and isolated a series of Ca2+-binding proteins by taking advantage of their Ca2+-dependent hydrophobic interaction with phenyl-Sepharose beads. The proteins identified by micro-sequencing analysis included calmodulin, neurocalcin, sorcin, annexin II and annexin V. Immunohistochemical analysis of the chick pineal sections revealed that both calmodulin and sorcin are expressed in the follicular and parafollicular pinealocytes. On the other hand, neurocalcin was expressed in a few neuron-like cells located predominantly in the parafollicular layer of the pineal follicle. These results suggest that calmodulin and sorcin may contribute to cellular functions in the chick pinealocytes.

The Morpho‐anatomy and Histology of the Pineal Complex in a Major Indian Carp, Catla catla: Identification of the Pineal Photoreceptor Cells and Their Responsiveness to Constant Light and Constant Darkness During Different Phases of the Annual Reproductive Cycle

In contrast to mammals in which the pineal gland is a discrete structure situated dorsally in the brain, the “pineal gland” in teleost fishes is composed of a number of separate but connected constituent parts, collectively described as the “pineal complex.” In this paper, we have described the pineal complex in a common Indian carp, Catla catla, which exhibits an annual reproductive cycle. Attempts have been made to (a) provide an in‐depth description of the structure of the pineal complex; and (b) identify the photoreceptor cells of the pineal, by exposing the animals to constant light (LL) and constant darkness (DD). Furthermore, we examined any possible influence of the reproductive status of the fish on the responsiveness of the pineal photoreceptor cells in C. catla following exposure to LL and DD. To this end, a total of four experiments were carried out during the four different phases of the annual reproductive cycle that is characteristic of this species. Each of these four experiments was carried out for a period of 30 days after which the fishes were sacrificed, different parts of the pineal complex were dissected out, and processed for histological and karyometric studies. Our results showed that the pineal complex in this species is composed of three separate but connected parts, (a) an end vesicle (EV); (b) a dorsal sac (DS); and (c) a long and thin pineal stalk (PS) that attaches the EV to the DS. Detailed karyometric and histo‐morphologic studies following exposure of the animals to DD and LL showed that constant darkness led to a stimulatory effect on the pineal photoreceptor cells of the EV as evident from a significant increase in the nuclear diameter. In contrast, the nuclear diameter of the photoreceptor cells in animals subjected to constant light showed a significant reduction. Furthermore, the observed cellular changes in the EV of fish exposed either to LL or DD were independent of the stage of the gonadal cycle. The apparent lack of any cellular responses either in the PS, or in the DS, following exposure to LL and DD, suggests that in C. catla the photoreceptor cells are located only within the epithelial lining of the EV and that these cells respond in a manner similar to mammalian pinealocytes when subjected to comparable photoperiod‐induced experimental conditions.

松果体細胞における光依存的な転写調節の分子メカニズム

松果体細胞における光依存的な転写調節の分子メカニズム

The Structure of the Pineal Complex in a Common Indian Teleost, Catla catla: Evidence for Pineal‐Induced Inhibition of Testicular Function Within an Annual Reproductive Cycle

The structure of the pineal complex and the annual reproductive cycle in a major Indian carp, Catla catla, were investigated in the present study. Additionally, given the well‐known inhibitory effects of the pineal on reproductive function in mammals, attempts were made to investigate whether or not the pineal exerts an inhibitory influence on reproductive function in this piscine species as well. Sexually adult animals were utilized in all experiments. The cytomorphology of the pineal complex and a number of parameters for testicular function—such as testicular cytology, serum testosterone levels, and testicular activities of two steroidogenic enzymes, 17β‐hydroxysteroid dehydrogenase (17β‐HSD) and Δ5‐3β‐hydroxysteroid dehydrogenase (Δ5‐3β‐HSD) were examined over a period of two years. Our studies showed that the pineal complex in this species consists of three separate but distinctly connected components: (a) an end vesicle (EV); (b) a long pineal stalk (PS); and (c) a dorsal sac (DS). Of these, the epithelial lining of the EV consists of cells that have rounded vesicular nuclei and long apical cytoplasmic processes that reach the lumen, features suggestive of pineal photoreceptor cells. The cells of the PS have some similarity with those of the EV, while DS cells appear columnar and ciliated. With regard to gonadal activity, testicular germ cell profiles revealed that this species has four distinct phases during the annual reproductive cycle: (a) preparatory (January–April); (b) pre‐spawning (May–June); (c) spawning (July); and (d) post‐spawning (August–December). During the spawning phase (July), seminiferous tubular diameter, percentage of late spermatids within seminiferous tubules, and serum testosterone levels showed the highest values compared to those obtained in most of the other phases of the reproductive cycle. Also in July, along with peak serum testosterone levels, the activities of 17β‐HSD and Δ5‐3β‐HSD were at their highest levels. In a correlation between the pineal cytology and testicular functional status, it was noted that both the nuclear diameter and the apical cytoplasmic projections of the EV photoreceptor cells showed a significant reduction, thus suggesting a reduced synthetic activity, during the month of July, the spawning phase of the reproductive cycle. In contrast, the same features of the EV cells during the other phases of the reproductive cycle showed an increased cellular and metabolic activity—a time when the gonads were less active and in a quiescent stage. These data suggest an inhibitory role of the pineal on gonadal function and thus provide additional credence to the concept that, as in higher mammals, there exists an inverse relationship between the pineal activity and gonadal function in teleost fishes as well.

Melatonin suppresses NO-induced apoptosis via induction of Bcl-2 expression in PGT-beta immortalized pineal cells.

In the present study, we investigated whether melatonin would prevent nitric oxide (NO)-induced apoptotic death of PGT-beta immortalized pineal cells. To examine the protective effect of melatonin, cytotoxicity assay, DNA fragmentation analysis, caspase-3 activity assay, and Western blotting for caspase-3 and poly(ADP-ribose) polymerase (PARP) were performed. Treatment of cells with S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, was shown to induce apoptotic cell death in a dose-dependent manner, and pretreatment with melatonin (0.1 mm) attenuated the occurrence of NO-induced apoptotic cell death. DNA fragmentation in response to NO was also arrested by melatonin. Caspase-3 activity induced by NO was decreased with melatonin treatment. Furthermore, the active fragments of caspase-3 and PARP were almost completely absent following exposure to melatonin. To elucidate the protective mechanisms of action of melatonin, Western blot analyses for Bcl-2 expression and cytochrome c release were carried out. Pretreatment with melatonin (0.1 mm) induced the expression of Bcl-2 and suppressed the release of cytochrome c into the cytosol, thereby arresting NO-induced apoptotic cell death. These results suggest that the antiapoptotic effect of melatonin is associated with induction of Bcl-2 expression in PGT-beta cells, which in turn blocks caspase-3 activation and inhibits cytochrome c release into the cytosol.

Opsin-G11-Mediated Signaling Pathway for Photic Entrainment of the Chicken Pineal Circadian Clock

Light is a major environmental signal for entrainment of the circadian clock, but little is known about the intracellular phototransduction pathway triggered by light activation of the photoreceptive molecule(s) responsible for the phase shift of the clock in vertebrates. The chicken pineal gland and retina contain the autonomous circadian oscillators together with the photic entrainment pathway, and hence they represent useful experimental models for the clock system. Here we show the expression of G11alpha, an alpha subunit of heterotrimeric G-protein, in both tissues by cDNA cloning, Northern blot, and Western blot analyses. G11alpha immunoreactivity was colocalized with pinopsin in the chicken pineal cells and also with rhodopsin in the outer segments of retinal photoreceptor cells, suggesting functional coupling of G11alpha with opsins in the clock-containing photosensitive tissues. The physical interaction was examined by coimmunoprecipitation experiments, the results of which provided evidence for light- and GTP-dependent coupling between rhodopsin and G11alpha. To examine whether activation of endogenous G11 leads to a phase shift of the oscillator, Gq/11-coupled m1-type muscarinic acetylcholine receptor (mAChR) was ectopically expressed in the cultured pineal cells. Subsequent treatment of the cells with carbamylcholine (CCh), an agonist of mAChR, induced phase-dependent phase shifts of the melatonin rhythm in a manner very similar to the effect of light. In contrast, CCh treatment induced no measurable effect on the rhythm of nontransfected (control) cells or cells expressing G(i/o)-coupled m2-type mAChR, indicating selectivity of the G-protein activation. Together, our results demonstrate the existence of a G11-mediated opsin-signaling pathway contributing to the photic entrainment of the circadian clock.

Morphometric analysis of photoreceptive, neuronal and endocrinal cell differentiation of avian pineal cells: an in vitro immunohistochemical study on the developmental transition from neuronal to photo-endocrinal property.

Little is known about the developmental origin, determination and differentiation of different pineal immunoreactive cells in the avian group, and an experimental establishment is then required to explain the differentiation of cell types (i.e. photosensory, neural and secretory types). The present in vitro study suggests that the avian pineal organ is made up of multiple types of cells with different immunoreactivity at the ontogenic state (from embryonic day 9 to day 14), before it acquires the final photoendocrinal nature of the mature state. The morphometric analysis suggests that the developmental changes in the morphology of the quail pinealocytes appear to represent a condensed expression of the phylogenic development in the ontogeny. Several types of immunoreactive cells from a neuronal line were suppressed with maturation of developing pineal glands, while other cell types such as photoreceptive and endocrinal lines became more prominent. The melatonin level in the culture medium presented a high value up to 72 hr of culture, followed by a decrease as well as dampening of the level at the end of the culture possibly because the cultures were maintained in dark. The results of the present study, a combined analysis of morphometry and RIA, open a new line for research into the pineal development and cell differentiation.

A Negative Regulatory Element Required for Light-Dependentpinopsin Gene Expression

In vertebrates, a variety of light-stimulated genes are distributed in the retina, the pineal gland, and the suprachiasmatic nucleus, but a cis-element(s) responsible for the light-dependent transcriptional regulation is left unexplored. Focusing on the pinopsin gene, a light-stimulated gene in the chick pineal gland, we performed a transcriptional analysis in the primary culture of the chick pineal cells that were transiently transfected with a luciferase reporter gene fused with various lengths of the 5' upstream region of the pinopsin gene. Light-dependent enhancer activity was detectable in the construct with the upstream region between -1156 and +31. Introduction of mutations within the 18 bp sequence at positions -1103 to -1086 (TGGCACGTGGGGTTCCTC), including a CACGTG E-box sequence, elevated the transcriptional activity in the dark and thereby abrogated the light dependency, suggesting that the 18 bp sequence is essential for a reduction of the transcriptional activity in the dark. In an electrophoretic mobility-shift assay, we identified a pineal nuclear factor(s) capable of binding to the 18 bp element in a sequence-specific manner. When a 49 bp fragment (-1122 to -1074) including the 18 bp sequence was placed upstream of the simian virus 40 promoter, the transcriptional activity was dramatically suppressed regardless of light conditions in the chick pineal cells, and a more pronounced repression was observed in nonpineal/nonphotosensory LMH and NIH 3T3 cells. These results suggest that the 18 bp element in the pinopsin promoter constitutes the binding site of a ubiquitous factor that serves for the transcriptional repression that is required, although not sufficient, for the light-dependent expression of pinopsin gene in the chick pinealocytes.

Norepinephrine triggers Ca2+-dependent exocytosis of 5-hydroxytryptamine from rat pinealocytes in culture.

5-hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from l-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT2 receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca2+-dependent release of 5-HT, which was blocked by phentolamine, an alpha antagonist, but not by propranolol, a beta antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H+-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca2+-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated l-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca2+-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in melatonin release via the specific receptor PACAP-r1, but not in the circadian oscillator, in chick pineal cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates melatonin release from pineal cells and modulates glutamatergic regulation of the suprachiasmatic circadian clock in rodents. We investigated whether PACAP is involved in melatonin release and the circadian oscillation system in chick pineal cells, and if so, whether its effects are mediated by the PACAP-specific receptor (PACAP-r1) or the vasoactive intestinal polypeptide (VIP) receptor. Chick pineal cells were maintained for 4 days under a 12-h light/dark cycle, and thereafter in constant darkness. In the dose-range 10 −10 to 10 −6 M, PACAP increased melatonin release dose-dependently during the 12-h light period on day 3 of culture, and the degree of stimulation was greater than that produced by VIP. VIP receptor antagonists only slightly inhibited PACAP-stimulated melatonin release. Simultaneous addition of VIP and PACAP produced almost additive melatonin release. Under constant dark conditions, 6-h pulses of PACAP started at zeitogaber times (ZT) 15, 21, 3 and 9 h in separate groups of pineal cells did not cause any phase shift in their melatonin rhythm. In addition, PACAP did not affect the light-induced phase advance (ZT 15 h) and delay (ZT 9 h) in melatonin rhythms. The expression of mRNA for the PACAP-r1 (including its splicing variant with a hop cassette) was observed in chick pineal cells. These results suggest that PACAP participates in melatonin release, but not in the circadian oscillator system, via the specific receptor PACAP-r1 in chick pineal cells.

Nonvisual photoreceptors of the deep brain, pineal organs and retina.

The role of the nonvisual photoreception is to synchronise periodic functions of living organisms to the environmental light periods in order to help survival of various species in different biotopes. In vertebrates, the so-called deep brain (septal and hypothalamic) photoreceptors, the pineal organs (pineal- and parapineal organs, frontal- and parietal eye) and the retina (of the "lateral" eye) are involved in the light-based entrain of endogenous circadian clocks present in various organs. In humans, photoperiodicity was studied in connection with sleep disturbances in shift work, seasonal depression, and in jet-lag of transmeridional travellers. In the present review, experimental and molecular aspects are discussed, focusing on the histological and histochemical basis of the function of nonvisual photoreceptors. We also offer a view about functional changes of these photoreceptors during pre- and postnatal development as well as about its possible evolution. Our scope in some points is different from the generally accepted views on the nonvisual photoreceptive systems. The deep brain photoreceptors are hypothalamic and septal nuclei of the periventricular cerebrospinal fluid (CSF)-contacting neuronal system. Already present in the lancelet and representing the most ancient type of vertebrate nerve cells ("protoneurons"), CSF-contacting neurons are sensory-type cells sitting in the wall of the brain ventricles that send a ciliated dendritic process into the CSF. Various opsins and other members of the phototransduction cascade have been demonstrated in telencephalic and hypothalamic groups of these neurons. In all species examined so far, deep brain photoreceptors play a role in the circadian and circannual regulation of periodic functions. Mainly called pineal "glands" in the last decades, the pineal organs actually represent a differentiated form of encephalic photoreceptors. Supposed to be intra- and extracranially outgrown groups of deep brain photoreceptors, pineal organs also contain neurons and glial elements. Extracranial pineal organs of submammalians are cone-dominated photoreceptors sensitive to different wavelengths of light, while intracranial pineal organs predominantly contain rod-like photoreceptor cells and thus scotopic light receptors. Vitamin B-based light-sensitive cryptochromes localized immunocytochemically in some pineal cells may take part in both the photoreception and the pacemaker function of the pineal organ. In spite of expressing phototransduction cascade molecules and forming outer segment-like cilia in some species, the mammalian pineal is considered by most of the authors as a light-insensitive organ. Expression of phototransduction cascade molecules, predominantly in young animals, is a photoreceptor-like characteristic of pinealocytes in higher vertebrates that may contribute to a light-percepting task in the perinatal entrainment of rhythmic functions. In adult mammals, adrenergic nerves--mediating daily fluctuation of sympathetic activity rather than retinal light information as generally supposed--may sustain circadian periodicity already entrained by light perinatally. Altogether three phases were supposed to exist in pineal entrainment of internal pacemakers: an embryological synchronization by light and in viviparous vertebrates by maternal effects (1); a light-based, postnatal entrainment (2); and in adults, a maintenance of periodicity by daily sympathetic rhythm of the hypothalamus. In addition to its visual function, the lateral eye retina performs a nonvisual task. Nonvisual retinal light perception primarily entrains genetically-determined periodicity, such as rod-cone dominance, EEG rhythms or retinomotor movements. It also influences the suprachiasmatic nucleus, the primary pacemaker of the brain. As neither rods nor cones seem to represent the nonvisual retinal photoreceptors, the presence of additional photoreceptors has been supposed. Cryptochrome 1, a photosensitive molecule identified in retinal nerve cells and in a subpopulation of retinal photoreceptors, is a good candidate for the nonvisual photoreceptor molecule as well as for a member of pacemaker molecules in the retina. When comparing various visual and nonvisual photoreceptors, transitory, "semi visual" (directional) light-perceptive cells can be detected among them, such as those in the parietal eye of reptiles. Measuring diffuse light intensity of the environment, semivisual photoreceptors also possess some directional light perceptive capacity aided by complementary lens-like structures, and screening pigment cells. Semivisual photoreception in aquatic animals may serve for identifying environmental areas of suitable illumination, or in poikilotermic terrestrial species for measuring direct solar irradiation for thermoregulation. As directional photoreceptors were identified among nonvisual light perceptive cells in the lancelet, but eyes are lacking, an early appearance of semivisual function, prior to a visual one (nonvisual --> semivisual --> visual?) in the vertebrate evolution was supposed.

Differential labeling of the pinealocytes and pineal interstitial cells by a series of monoclonal antibodies to human pineal body.

Ten hybridomas producing monoclonal antibodies against pineal antigens were established by hybridizing mouse myeloma cells with spleen cells from BALB/c mice immunized with human pineal body homogenate. Seven antibodies immunohistochemically reacted with the pinealocytes and three reacted with the pineal interstitial cells. According to the antibodies applied, the pinealocytes were immunostained in a variable manner; granularly with PP1, PP4 and PP6 antibodies, diffusely with PP2 and PP5, membraneously with PP3, and apically with PP7. The PI1 and P12 antibodies immunolabeled most of the interstitial cells, but PX1 immunolabeled only a small population of these cells. An immunoblotting study indicated that single or multiple polypeptides of the pineal homogenate constituted the antigen epitopes for these antibodies. Most antibodies also immunoreacted with several cell types of the extra-pineal tissues, thus these antibodies are not specific to the human pineal body. This series of monoclonal antibodies should be available for immunohistochemical studies involving the normal and pathological human pineal body.

CDNA array analysis of pineal gene expression reveals circadian rhythmicity of the dominant negative helix-loop-helix protein-encoding gene, Id-1.

The pineal gland is a major output of the endogenous vertebrate circadian clock, with melatonin serving as the output signal. In many species, elevated nocturnal melatonin production is associated with changes in pineal gene expression. In the current study, cDNA array analysis was used in an attempt to identify additional genes that exhibit day/night differential expression in the rat pineal gland. This revealed 38 candidate genes, including Id-1 (inhibitor of DNA binding and differentiation). Id-1 encodes a helix-loop-helix (HLH) protein that lacks a basic DNA binding domain and could affect pineal physiology via a dominant negative trans-acting regulatory activity. For this reason Id-1 was selected for further analysis. Id-1 was expressed in a major population of pineal cells and the Id-1 protein was associated with a nuclear complex. The levels of Id-1 mRNA and protein exhibit approximately six-fold day/night rhythms. In contrast, the related genes Id-2 and Id-3 do not exhibit marked day/night differences in pineal expression. Rhythmic Id-1 expression is primarily limited to a C-terminally extended splice variant of Id-1, which would restrict the functional output of the rhythm to protein binding partners of this isoform of Id-1. Our findings add to the body of evidence indicating that transcriptional regulators play a role in neuroendocrine rhythms, and extend this by introducing the concept of a dominant negative HLH involvement. The rhythm in Id-1 in the pineal gland provides an experimental opportunity to identify Id-1-binding partners which may also be involved in Id-1 activity in other functional contexts.

Glutamate receptor subunit delta2 is highly expressed in a novel population of glial-like cells in rat pineal glands in culture.

The mammalian pineal gland uses L-glutamate as an intercellular chemical transmitter to regulate negatively melatonin synthesis. To receive glutamate signals, pinealocytes express at least three kinds of glutamate receptors: metabotropic receptor types 3 and 5 and an ionotropic receptor, GluR1. In this study, we examined whether or not the fourth class of ionotropic receptor, delta, which is known for its nondefinitive molecular function and its unique expression pattern in brain, is expressed in pineal gland. RT-PCR analyses with specific probes indicated the expression of mRNA of delta2 but not that of delta1 in pineal gland and cultured pineal cells. Western blotting analysis with polyclonal antibodies specific to the carboxyl-terminal region of the delta2 receptor recognized a single 110-kDa polypeptide of cerebellar membranes and specifically immunostained Purkinje cells. The delta2 antibodies recognized a 110-kDa polypeptide of pineal membranes and specifically immunostained huge glial-like cells with the occasional presence of several long, branching processes in a pineal cell culture. delta2 is not uniformly distributed throughout the cells and is relatively abundant at the periphery of the cell bodies and long processes, where the terminals of synaptophysin-positive processes of pinealocytes, a site for glutamate secretion, are frequently present. The delta2-positive cells constitute a very minor population among total pineal cells (approximately 0.03%). Double immunolabeling with delta2 antibodies and antibodies against marker proteins for pineal interstitial cells clearly distinguishes delta2-positive pineal cells and other known interstitial cells, including glial fibrillary acidic protein- or vimentin-positive glial-like cells. These results indicated that the delta2 glutamate receptor is expressed in a novel subpopulation of pineal glial-like cells in culture and suggest the presence of a glutamate-mediated intercellular signal transduction mechanism between pinealocytes and delta2-expressing cells. The pineal cells may provide a good experimental system for studies on the function of glutamate receptor delta2.

Photoendocrine signal transduction in pineal photoreceptors of the trout. Role of cGMP and nitric oxide.

This study describes the presence and distribution of cGMP-immunoreactivity and of the nitric oxide (NO) synthesizing enzyme, NO synthase (NOS), as demonstrated by use of the NADPH-diaphorase technique in directly light sensitive pineal organ of the trout. Cyclic GMP immunohistochemistry revealed immunoreactivity in pineal photoreceptor cells that were identified by double-labeling with S-antigen, whereas NADPH-positive structures were located adjacent to these photoreceptor cells. Since NO is known to stimulate synthesis of cGMP, these results indicate a role for NO in pineal function, e.g. in cGMP related events in the phototransduction process as well as in the light-dark control of melatonin synthesis.

Effects of vasoactive intestinal peptide and histamine on melatonin and cAMP production in chick embryo pineal cells.

The avian pineal gland, which seems to be a major source of circulating melatonin, contains a circadian oscillator that controls the rhythm of melatonin synthesis and release (1). Melatonin production is rhythmic in chick pineal cells. The key regulator of melatonin synthesis is cAMP. Agents which increase cAMP levels, such as vasoactive intestinal peptide (VIP), increase melatonin production (2). One of the widely reported actions of histamine (HA) is stimulation of cAMP production. The effect of HA on cAMP synthesis in the chick pineal gland was studied under in vivo and in vitro conditions and the gland was found to be highly sensitive to this amine

Chicken pineal Cry genes: light-dependent up-regulation of cCry1 and cCry2 transcripts

Vertebrate cryptochrome homologs (CRYs) are negative regulators for the transcription/translation-based autoregulatory feedback loop of the circadian clock. In this study we identified two Cry genes in the chicken, cCry1 and cCry2, which are expressed in the pineal gland. Messenger RNA levels of both cCry1 and cCry2 displayed circadian oscillation in cultured pineal cells under light/dark and constant darkness conditions. Noticeably, their mRNA levels during the light period were significantly higher than those in the dark, indicating light-dependent up-regulation of the two Cry genes mediated by photoreceptor(s) intrinsic to the chick pineal cells. These cCRYs inhibited E-box element-dependent cBMAL1/2-cCLOCK-induced transcription, suggesting that the chick pineal circadian oscillator is composed of molecules that are functionally similar to those of mammals but are subject to light-regulation distinct from the mammalian clockwork.

Dopamine induces intracellular Ca 2+ signals mediated by α 1B-adrenoceptors in rat pineal cells

We have studied the functional interaction of dopamine with α 1-adrenoceptor subtypes by measuring intracellular Ca 2+ levels in pineal cells, a cell type where adrenoceptors are well characterized. We show that dopamine induces transient intracellular Ca 2+ signals in only 70% of cells responding to phenylephrine. Dopamine-induced Ca 2+ signals desensitise faster than Ca 2+ transients elicited with phenylephrine and are selectively blocked by desipramine, imipramine, and α 1B-adrenoceptor antagonists. These results suggest that dopamine induced Ca 2+ signals are mainly due to the activation of one subtype of α-adrenoceptor, the α 1B.

Expression and role of phosphodiesterase 6 in the chicken pineal gland.

The chicken pineal gland is directly photosensitive, with light causing an inhibition of melatonin synthesis. A possible role of phosphodiesterase 6 (PDE6, the primary effector of retinal phototransduction) in mediating this response was investigated. RT-PCR, DNA sequencing and northern blots revealed the presence of RNA encoding both catalytic and regulatory subunits of PDE6 in the chicken pineal gland. Both rod and cone forms of PDE6 subunits mRNA were detected. The concentration of the transcripts encoding PDE6 catalytic subunits peaked at night. Western blot analysis of chicken pineal proteins with an antibody directed against the catalytic subunits of bovine rod PDE6 identified a single immunoreactive protein of 97 kDa. Anion exchange chromatography of chicken pineal soluble proteins revealed a peak of PDE6 activity that accounted for about 30% of cyclic GMP-hydrolysis. In cultured chick pineal glands, arylalkylamine N-acetyltransferase (AA-NAT), the rate-limiting enzyme of melatonin synthesis, was protected from inhibition by light when selective PDE5/6 inhibitors (zaprinast, DMPPO) were added to the culture medium. PDE5/6 inhibitors did not affect AA-NAT activity in the dark. In contrast, a general PDE inhibitor (IBMX) increased AA-NAT in a light-independent manner. Together, the data indicate that rod and cone forms of PDE6 are expressed in chick pineal cells and that this enzyme plays a role in the inhibition of melatonin synthesis by light.