Showing papers on "Pinealocyte published in 1999"

Retinopathy and attenuated circadian entrainment in Crx-deficient mice.

Abstract: Crx, an Otx-like homeobox gene, is expressed specifically in the photoreceptors of the retina and the pinealocytes of the pineal gland. Crx has been proposed to have a role in the regulation of photoreceptor-specific genes in the eye and of pineal-specific genes in the pineal gland. Mutations in human CRX are associated with the retinal diseases, cone-rod dystrophy-2 (adCRD2; refs 3, 4, 5), retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), which all lead to loss of vision. We generated mice carrying a targeted disruption of Crx. Crx-/- mice do not elaborate photoreceptor outer segments and lacked rod and cone activity as assayed by electroretinogram (ERG). Expression of several photoreceptor- and pineal-specific genes was reduced in Crx mutants. Circadian entrainment was also affected in Crx-/- mice.

The pineal gland and melatonin: molecular and pharmacologic regulation.

Abstract: ▪ Abstract The pineal gland expresses a group of proteins essential for rhythmic melatonin production. This pineal-specific phenotype is the consequence of a temporally and spacially controlled program of gene expression. Understanding of pineal circadian biology has been greatly facilitated in recent years by a number of molecular studies, including the cloning of N-acetyltransferase, the determination of the in vivo involvement of the cAMP-inducible early repressor in the regulation of N-acetyltransferase, and the identification of a pineal transcriptional regulatory element and its interaction with the cone-rod homeobox protein. Likewise, appreciation the physiological roles of melatonin has increased dramatically with the cloning and targeted knockout of melatonin receptors. With these molecular tools in hand, we can now address more specific questions about how and why melatonin is made in the pineal at night and about how it influences the rest of the body.

Transcription Factors in Neuroendocrine Regulation: Rhythmic Changes in pCREB and ICER Levels Frame Melatonin Synthesis

Abstract: Neurotransmitter-driven activation of transcription factors is important for control of neuronal and neuroendocrine functions. We show with an in vivo approach that the norepinephrine cAMP-dependent rhythmic hormone production in rat pineal gland is accompanied by a temporally regulated switch in the ratio of a transcriptional activator, phosphorylated cAMP-responsive element–binding protein (pCREB), and a transcriptional inhibitor, inducible cAMP early repressor (ICER). pCREB accumulates endogenously at the beginning of the dark period and declines during the second half of the night. Concomitant with this decline, the amount of ICER rises. The changing ratio between pCREB and ICER shapes the in vivo dynamics in mRNA and, thus, protein levels of arylalkylamine- N -acetyltransferase, the rate-limiting enzyme of melatonin synthesis. Consequently, a silenced ICER expression in pinealocytes leads to a disinhibited arylalkylamine- N -acetyltransferase transcription and a primarily enhanced melatonin synthesis.

A Novel Pineal Night-Specific ATPase Encoded by the Wilson Disease Gene

Abstract: We have identified a pineal night-specific ATPase (PINA), a novel splice variant of the ATP7B gene disrupted in Wilson disease (WD). PINA expression exhibits a dramatic diurnal rhythm in both pineal gland and retina with 100-fold greater expression at night than at day. PINA is expressed in pinealocytes and a subset of photoreceptors in adult rats and is transiently expressed in the retinal pigment epithelium and the ciliary body during retinal development. Nocturnal pineal expression of PINA is under the control of a suprachiasmatic nucleus clock mediated by superior cervical ganglion innervation of the pineal. In vitro, PINA expression in pineal cells can be stimulated by agents activating the cAMP signal transduction pathway. PINA is able to restore copper transport activity in Saccharomyces cerevisiae deficient in the homologous copper-transporting ATPase CCC2, suggesting that this protein may function as a copper transporter in rat pinealocytes. These studies suggest a potential role of rhythmic copper metabolism in pineal and/or retina circadian function.

Inducible cyclic AMP early repressor protein in rat pinealocytes: a highly sensitive natural reporter for regulated gene transcription.

Abstract: Rhythmic activity of arylalkylamine N-acetyltransferase (AANAT) determines melatonin synthesis in rat pineal gland. The transcriptional regulation of AANAT involves the activating and inhibiting transcription factors of the cyclic AMP (cAMP)-signaling pathway, cAMP response element-binding protein and inducible cAMP early repressor (ICER), respectively. Activation of this pathway is centered around norepinephrine, stimulating β1-adrenergic receptors, but various other transmitters can modulate melatonin biosynthesis. To compare the transcriptional impact of norepinephrine with that of other neurotransmitters on melatonin synthesis, we determined ICER protein levels in pinealocytes and, in parallel, hormone secretion. The dose-dependent inductions of ICER protein by norepinephrine, the β1-adrenergic receptor agonist isoproterenol, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and adenosine are correlated to regulatory dynamics in melatonin production. Importantly, ICER protein induction required lower ligand concentrations than the induction of melatonin biosynthesis. Although neuropeptide Y, glutamate, and vasopressin altered norepinephrine-stimulated hormone production without affecting ICER levels, the activation of voltage-gated cation channels increased ICER without affecting hormone synthesis. Sensitivity and versatility of ICER induction in pinealocytes make these neuroendocrine cells a valuable model system in which to study molecular interactions determining a regulated gene expression.

Cholinergic innervation and function in the mammalian pineal gland.

Abstract: Besides the noradrenergic sympathetic system originating from the superior cervical ganglion, a cholinergic innervation of the mammalian pineal gland has been studied over the past three decades. In 1961, it was shown that lesion of the parasympathetic greater superficial petrosal nerve of the monkey resulted in degeneration of nerve fibers in the pineal gland. This was supported by ultrastructural studies of nerve terminals within the pineal gland, demonstrating the presence of cholinergic terminals containing small clear transmitter vesicles. Biochemical studies further showed the presence of the enzyme acetylcholinesterase in several mammalian species. During the last decade, several advanced and more elaborate technologies have been developed, allowing pinealogists to establish the presence of cholinergic fibers and their receptors. Thus, choline acetyltransferase was shown in bovine pineal by immunohistochemistry. Muscarinic and nicotinic receptors were identified, characterized, and localized. Gene expression of receptors was visualized, and the receptor-mediated effector systems and functions were elucidated. Taken together, the present data suggest the presence of a cholinergic innervation of the mammalian pineal gland originating in peripheral parasympathetic ganglia. However, some of the neuronal projections to the pineal gland with origin in the brain (the central innervation) might also be cholinergic. The cholinergic nerve fibers enter the gland, where they are located both in the perivascular spaces and between the pinealocytes. Some of the terminals make synapses on pinealocytes or intrapineal neurons. The released acetylcholine from the terminals interacts with the receptors, then alters the cascade of receptor-mediated events, which results in decreased N-acetyltransferase enzyme activity, thus leading to decreased melatonin synthesis. This counterbalance mechanism between the sympathetic noradrenergic and the cholinergic systems maintains the homeostasis of pineal functions.

Ceramide inhibits L-type calcium channel currents in rat pinealocytes.

Abstract: In rat pinealocytes, ceramide can inhibit the KCl- and BayK 8644-mediated potentiation of cAMP and cGMP accumulation, suggesting that the L-type Ca2+ channel is a target of ceramide action. This was examined in the present study using intracellular Ca2+ measurement and patch-clamp studies. In fura-2-loaded pinealocytes, C2- and C6-ceramide inhibited the Ca2+ increase caused by BayK 8644 and KCl, but not that caused by norepinephrine, suggesting an inhibitory effect of ceramide on the L-type Ca2+ channels. Patch-clamp analysis confirmed that C2- and C6-ceramide, but not C2-dihydroceramide (the inactive analog) inhibited the L-type Ca2+ channel current. Furthermore, treatments known to increase cellular ceramide levels, including a glucosylceramide synthase inhibitor and sphingomyelinase, also inhibited this current. The inhibitory effect of ceramide on the current was attenuated by lavendustin A, a tyrosine kinase inhibitor, but not by H7, a serine/threonine kinase inhibitor. The effect of ceramide was mim...

Wavelength dependency of light-induced changes in rhythmic melatonin secretion from chicken pineal gland in vitro.

Abstract: Avian pineals show rhythmic, circadian melatonin secretion pattern also in vitro. The phase of this rhythm can be modified by changing the illumination. Reversed in vitro illumination reverses the phase of the circadian melatonin rhythm in two days. In the present study the effect of the wavelength on the speed of the phase shift of the melatonin rhythm of the explanted chicken pineals was investigated in a dynamic in vitro bioassay. Chicken pineals were placed into perifusion columns and the tissue was exposed to reversed illumination through various light filters. Changes in the melatonin release during 4 day long experiments were studied. Clear differences in the speed of the reversal of the melatonin rhythm were observed as a function of the transmission spectrum of the light filters. The shorter the transmitted wavelength, the more rapid the phase shift was found. These data are in good agreement with earlier studies showing that the chicken pineal photo-pigment, pinopsin, is a blue light sensitive molecule. Our observation reveals that the blue light sensitive pigment is not only present but also fully functional in controlling the circadian biological clock in the chicken pineal gland.

Neuropeptide Y increases intracellular calcium in rat pinealocytes.

Abstract: The pineal gland is mainly innervated by sympathetic fibres containing noradrenaline (NA) and neuropeptide Y (NPY). NA released at night stimulates melatonin synthesis via a beta1-adrenergic-induced increase in cyclic adenosine monophosphate (cAMP) concentration potentiated by an alpha1-adrenergic-induced increase in Ca2+ concentration. We previously showed that NPY acted on presynaptic Y2 receptors inhibiting NA release and on postsynaptic Y1 receptors stimulating melatonin synthesis. Here we used Fura-2 imaging to assess the effect of NPY on the intracellular Ca2+ concentration, [Ca2+]i, in cultured rat pineal cells. In 84% of cells, on average, 10 nM NPY induced a progressive rise of [Ca2+]i from its basal value of 102+/-3 nM to a plateau of 180+/-6 nM (n = 467 cells), which lasted the time of NPY application. This effect of NPY appeared dependent on extracellular Ca2+.

Peptidergic peripheral nervous systems in the mammalian pineal gland.

Abstract: The distribution and density of tyrosine hydroxylase (TH) and neuropeptide Y (NPY)-immunoreactive, sympathetic fibers and calcitonin gene-related peptide (CGRP)-, substance P (SP)-, and vasoactive intestinal polypeptide (VIP)-immunoreactive, non-sympathetic fibers in the pineal gland, the effects of superior cervical ganglionectomy (SCGX) on these fibers, and the location of their terminals in the pineal gland were compared between rodents and non-rodents. A dense network of TH/NPY-positive fibers is present all over the pineal gland. A less dense network of CGRP/SP- or VIP-positive fibers occurs in the whole pineal gland of non-rodents, but these fibers are usually confined to the superficial pineal gland in rodents. After SCGX, some TH/NPY-fibers remain only in the deep pineal gland in rodents, whereas considerable numbers of these fibers persist throughout the gland in non-rodents. Thus, the remaining fibers, probably originating from the brain, may be more numerous in non-rodents. Since CGRP-, SP- or VIP-immunoreactive fibers in the pineal capsule can be traced to those in the gland, and since these fibers are ensheathed by Schwann cells, it is concluded that these fibers belong to the peripheral nervous system. However, the existence of SP-positive central fibers cannot be denied in some species. In the superficial pineal gland of rodents, sympathetic terminals are mostly localized in perivascular spaces, whereas the parenchymal innervation by sympathetic fibers in the pineal gland is more dense in non-rodents than in rodents. Synapses between sympathetic nerve terminals and pinealocytes occur occasionally in non-rodents, but only rarely in the superficial pineal gland of rodents. The occurrence of the synapses may depend on the frequency of intraparenchymal sympathetic terminals.

Synaptic-like microvesicles in mammalian pinealocytes.

Abstract: The recent deciphering of the protein composition of the synaptic vesicle membrane has led to the unexpected identification of a compartment of electron-lucent microvesicles in neuroendocrine cells which resemble neuronal synaptic vesicles in terms of molecular structure and function. These vesicles are generally referred to as synaptic-like microvesicles (SLMVs) and have been most intensively studied in pancreatic β-cells, chromaffin cells of the adrenal medulla, and pinealocytes of the pineal gland. This chapter focuses on the present knowledge of SLMVs as now well-established constituents of mammalian pinealocytes. I review the results of morphological, immunocytochemical, and biochemical studies that were important for the characterization of this novel population of secretory vesicles in the pineal organ. The emerging concept that SLMVs serve as a device for intercellular communication within the pineal gland is outlined, and unanswered questions such as those pertaining to the physiological function and regulation of pineal SLMVs are discussed.

Neuropeptide Y in the mammalian pineal gland.

Abstract: The present review describes the anatomy of the neuropeptide (NPY)ergic innervation of the mammalian pineal gland with emphasis on the rat. The proNPY-molecule is post-translationally processed by a single cleavage to neuropeptide Y (NPY) and a C-terminal peptide of NPY (CPON). NPY is C-terminally amidated, and the amidation is essential for binding of NPY to its corresponding receptor(s). Since no proNPY has been detected in rat pineal extracts, it is considered that proNPY is immediately processed to its final products in the gland. In the rat, numerous NPY- and CPON-immunoreactive (ir) nerve fibers are present in the capsule of the superficial pineal gland and in the pineal parenchyma, mostly related to the connective tissue spaces and the vasculature of the gland, but also present between the pinealocytes. Furthermore, a substantial number of fibers was observed in the deep pineal gland, the pineal stalk, and the underlying epithalamus. Occasionally, NPY- or CPON-immunoreactive fibers were found adjacent to the stria medullaris and in the posterior commissure, which could be followed to the adjacent deep pineal gland. At the ultrastructural level, the NPY-immunoreactivity was confined in boutons containing large granular vesicles (100-200 nm) as well as small (40-60 nm) granular vesicles. Some terminals were located in very close apposition to the pinealocyte cell membrane. Terminals were identified in perivascular spaces, but synaptic contacts between the immunoreactive terminals and pinealocytes were never observed. These data show that NPY is highly concentrated in nerve fibers throughout the rat pineal complex. Double-fluorescence histochemistry using tyrosine hydroxylase as marker for catecholaminergic fibers and NPY revealed that nearly all NPYergic fibers co-stored tyrosine hydroxylase in the superficial pineal gland. A minor portion of both immunoreactivities was not colocalized. In accordance, about 65% of the neurons in the superior cervical ganglion contained both CPON and tyrosine hydroxylase. In bilateral superior cervical ganglionectomized rats, a few NPY-ir nerve fibers remained mostly in the pineal capsule, but few fibers were also found in the superficial pineal parenchyma. Contrarily, only a moderate decrease was observed in the number of immunoreactive fibers in the deep pineal gland, and no reduction was observed in the adjacent epithalamus. In the ganglionectomised rats, co-localisation of tyrosine hydroxylase and NPY in intrapineal nerve fibers was not observed either in the superficial pineal gland, nor in the deep pineal gland. These results together with the available literature show that NPY is a sympathetic transmitter, and its actions in the pineal gland are, therefore, associated with the well-documented roles of noradrenaline. Possible roles of NPY in pineal biochemistry and physiology are discussed.

Regulation of the L-type Ca2+ channel current in rat pinealocytes: role of basal phosphorylation

Abstract: In the present study, the role of phosphoprotein phosphatase in the regulation of L-type Ca2+ channel currents in rat pinealocytes was investigated using the whole-cell version of the patch-clamp technique. The effects of three phosphatase inhibitors, calyculin A, tautomycin, and okadaic acid, were compared. Although all three inhibitors were effective in inhibiting the L-type Ca2+ channel current, calyculin A was more potent than either tautomycin or okadaic acid, suggesting the involvement of phosphoprotein phosphatase-1. To determine the kinase involved in the regulation of these channels, cells were pretreated with H7 (a nonspecific kinase inhibitor), H89 (a specific inhibitor of cyclic AMP-dependent kinase), KT5823 (a specific inhibitor of cyclic GMP-dependent kinase), or calphostin C (a specific inhibitor of protein kinase C). Pretreatment with either H7 or calphostin C decreased the inhibitory effect of calyculin A on the L-type Ca2+ channel current. In contrast, pretreatment with H89 or KT5823 had no effect on the inhibition caused by calyculin A. Based on these observations, we conclude that basal phosphatase activity, probably phosphoprotein phosphatase-1, plays an important role in the regulation of L-type Ca2+ channel currents in rat pinealocytes by counteracting protein kinase C-mediated phosphorylation.

Functional expression of metabotropic glutamate receptor type 5 in rat pinealocytes.

Abstract: Mammalian pinealocytes, endocrine cells for melatonin, express class II metabotropic glutamate receptors (mGluRs), which are involved in negative regulation of melatonin synthesis through an inhibitory cAMP cascade. We investigated whether mGluRs other than class II receptors are expressed in rat pinealocytes. RT-PCR analysis and Northern blotting indicated that the mRNA of mGluR5, a class I receptor, was present in pineal glands. Quisqualate and 1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD), class I receptor agonists, increased the intracellular [Ca2+] of fura-2 loaded cultured pinealocytes in the absence of extracellular Ca2+ which is blocked by methylcarboxyphenylglycine, a class I receptor antagonist. These results suggest that mGluR5 is functionally expressed in pinealocytes and triggers Ca2+ efflux from intracellular stores.

Pharmacological characterization of β2-adrenoceptor in PGT-β mouse pineal gland tumour cells

Abstract: 1. The adrenoceptor in a mouse pineal gland tumour cell line (PGT-beta) was identified and characterized using pharmacological and physiological approaches. 2. Adrenaline and noradrenaline, adrenoceptor agonists, stimulated cyclic AMP generation in a concentration-dependent manner, but had no effect on inositol 1,4,5-trisphosphate production. Adrenaline was a more potent activator of cyclic AMP generation than noradrenaline, with half maximal-effective concentrations (EC50) seen at 175+/-22 nM and 18+/-2 microM for adrenaline and noradrenaline, respectively. 3. The addition of forskolin synergistically stimulated the adrenaline-mediated cyclic AMP generation in a concentration-dependent manner. 4. The pA2 value for the specific beta2-adrenoceptor antagonist ICI-118,551 (8.7+/-0.4) as an antagonist of the adrenaline-stimulated cyclic AMP generation were 3 units higher than the value for the betaI-adrenoceptor antagonist atenolol (5.6+/-0.3). 5. Treatment of the cells with adrenaline and forskolin evoked a 3 fold increase in the activity of serotonin N-acetyltransferase with the peak occurring 6 h after stimulation. 6. These results suggest the presence of beta2-adrenoceptors in mouse pineal cells and a functional relationship between the adenylyl cyclase system and the regulation of N-acetyltransferase expression.

Effects of near-ultraviolet (UV-A) light on melatonin biosynthesis in vertebrate pineal gland.

Abstract: The effects of near-ultraviolet (UV-A) irradiation on nocturnal activity of serotonin N-acetyltransferase (NAT; a key regulatory enzyme in melatonin biosynthesis) in the pineal gland of the rat and chick were investigated. Exposure of the animals to UV-A during the 4th or 5th hour of the dark phase of the 12:12 h light-dark (LD) cycle suppressed the night-driven NAT activity in a time-dependent manner, the effects being generally more pronounced in rats than in chicks. The UV-A-evoked suppression of the nocturnal NAT activity was completely restored within 2 h (chicks) or 3 h (rats) in animals which, after irradiation, were returned to darkness. When a short UV-A pulse was applied to the animals after midnight, it induced a decrease in the enzyme activity in both species; yet, the effect was readily reversible only in chicks. The results presented here, as well as other data, demonstrate that UV-A light is a powerful signal affecting the pineal melatonin-generating system both in mammals and avians, and that the involved mechanisms may differ in the tested species.

The age-related quantitative ultrastructural changes in pinealocytes of gerbils.

Abstract: OBJECTIVES. Relatively few ultrastructural studies of the pineal gland of aging animals have been published. The pineal gland of the gerbil is especially interesting in respect to aging because of its progressive calcification with age, and this species has been considered as an excellent model for research on aging. Therefore, the aim of the present study was to examine the quantitative ultrastructure of pinealocytes of the gerbil in three different age groups. METHODS. Three groups of animals were included in the study: 1-month-old, 3-month-old, and 14-month-old. Cross-sectional areas of the pinealocyte and its nucleus and relative volumes of the following cell organelles: mitochondria, lysosomes, Golgi apparatus, granular endoplasmic reticulum and calcareous concretions as well as the number of dense-core vesicles and "synaptic" ribbons were analyzed. RESULTS. No age-dependent changes were observed in the size of pinealocytes and their nuclei. The relative volume of mitochondria and the number of dense-core vesicles increased progressively with age, and that of lysosomes was lowest in the 1-month-old animals and increased at age of 3 and 14 months, whereas a decrease in the relative volume of granular endoplasmic reticulum was observed in 3- and 14-month-old gerbils in comparison with 1-month-old animals. No difference was observed in relative volume of Golgi apparatus and in the number of "synaptic" ribbons. The most striking change was observed in the formation of calcareous concretion within the pineal with age. The pineal gland of 1-month-old gerbils was essentially devoid of these structures, their number and size in 3-month-old animals were moderate, and increased dramatically in 14-month-old animals. CONCLUSION. The ultrastructural features of the gerbil pinealocyte in all examined age groups point to high metabolic activity of these cells.