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Transcriptional regulation of hydroxyindole O-methyltransferase in the chicken pineal gland: day/night changes and long-term effects of light and darkness.

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.

Acetylserotonin O-Methyltransferase↗

Cells expressing preproenkephalin mRNA in the rat pineal gland are not serotonin-producing pinealocytes: evidence using in situ hybridization combined with immunocytochemistry for serotonin.

1. Preproenkephalin (PPEnk) mRNA expressing cells have been identified in rat pineal gland using radioactive in situ hybridization histochemistry. 2. Approximately 7% of the cells in the pineal gland (7.5 +/- 0.86, mean +/- 95% CI) express PPEnk mRNA. These cells are distributed throughout the pineal as either scattered single cells or small groups of cells with large round or oval nuclei. 3. Using in situ hybridization combined with ABC immunocytochemistry for serotonin (5-HT) in the same pineal sections, the PPEnk mRNA labeling cells are found not to be serotonin-immunoreactive cells. These data indicate that the PPEnk mRNA is expressed in a certain discrete subpopulation of cells in the rat pineal gland and these cells are not serotonin-producing pinealocytes. 4. The physiologic role of PPEnk-derived peptides in the pineal remains unknown. It is possible that these peptides either are synthesized and secreted as hormones or act as pineal paracrine signals.

Animals↗

Melatonin secretion from organ-cultured pineal glands of rats: modulation by gonadectomy and gonadotropin-releasing hormone agonist administration.

The objective of the study was to evaluate the effect of pretreatments such as gonadectomy in male and female rats, and gonadotropin-releasing hormone agonist (GnRHa) administration in female rats, on levels of secretion of melatonin, using an organ culture of pineal glands. Gonadectomy 2 weeks before the animal was killed increased the amount of melatonin secreted into the medium by the pineal glands of female rats but not of male rats. The increase in in vitro melatonin secretion after ovariectomy in female rats was prevented by estrogen replacement. Ovariectomy 3 and 4 weeks before death also significantly increased the amount of melatonin secretion. Administration of GnRHa 2 weeks before decapitation significantly decreased serum estradiol concentrations and significantly increased melatonin secretion by the pineal glands of female rat. GnRHa administration 3 or 4 weeks before decapitation also significantly decreased serum estradiol concentrations, but did not increase pineal secretion of melatonin. The results indicate that ovariectomy increases melatonin secretion from organ-cultured pineal glands and that this increase is suppressed by estrogen in adult female rats. In contrast, orchiectomy in male rats does not influence in vitro secretion of melatonin. These results suggest that the GnRH-gonadotropin system may participate in the regulation of pineal melatonin secretion in adult female rats.

Animals↗

Characterization of a [3H]glutamate binding site in rat pineal gland: enhanced affinity following superior cervical ganglionectomy.

Glutamate, an excitatory neurotransmitter/neuromodulator involved in cell-to-cell communication within the central nervous system, is now believed to play a role in neuroendocrine function. In this study we describe a single, saturable, stereospecific, and temperature-, time-, and pH-dependent binding site for glutamate in the pineal gland of the rat (Kd = 612 +/- 23 nM, Bmax = 3.17 +/- 0.33 pmol/mg protein). After removal of the sympathetic innervation to the pineal gland, [3H]glutamate binding displayed a higher apparent affinity (Kd = 412 +/- 28 nM) (P < 0.05) without a change in binding site number (Bmax = 3.60 +/- 0.24 pmol/mg protein). No difference in [3H]glutamate binding site number was observed in pineal glands obtained from animals sacrificed during the middle of the light and dark periods. These data suggest a possible modulatory role for a glutamate binding site in pineal gland function.

Animals↗

Daily profiles of N-acetyltransferase measured at a single time in rat pineal glands, retinas, and Harderian glands.

Serotonin N-acetyltransferase activity (NAT) exhibited a daily cycle in light:dark (LD) 14:10 when it was measured in pineal glands taken from rats killed at a sequence of time points. The ratio of peak subjective night NAT to minimum subjective day NAT was 10.9/0.3 nmol per pineal gland per hour. When the rats were placed in constant dark the rhythm persisted (8.2/0.02). When the rats were placed in constant light the rhythm persisted with markedly attenuated amplitude (0.6/0.02). We also measured NAT profiles in rat pineal glands, Harderian glands, and retinas with alternative methods. We kept rats on six LD 14:10 light-dark cycles with lights-out beginning at midnight, 2 AM, 4 AM, 6 AM, 8 AM, or 10 AM and killing all the rats at one time point, 10 AM. We examined the NAT time profiles 4, 8, and 11 days following placement of the rats in the phase-shifted cycles. In addition, we measured the NAT profile in LD 2:22 and LD 22:2 by keeping the rats on twelve cycles for 11 days and killing all the rats at one time. Pineal NAT exhibited a rhythm in all the cycles: peak-dark/nadir-light values (nmol product per gland per hour) were 15.6/0.1 in LD 14:10, controls killed at successive time points. The ratios for the profiles obtained using the one time point procedure were 16.7/0.1 in LD 14:10 8.5/0.2 in LD 22.2, and 12.9/0.2 in LD 2.22. Increasing the photoperiod reduced the time to the NAT peak.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗

Timekeeping by the pineal gland.

N-Acetyltransferase, an enzyme involved in melatonin production in the pineal gland, exhibits a circadian rhythm in chickens with peak values in the dark-time and low values during the light-time, commencing at lights-on. When pineal glands of chickens killed during the dark-time (with high N-acetyltransferase activity) were organ-cultured, there was a decline in enzyme activity to light-time values. Regardless of the time of the dark at which the chickens were killed, the enzyme activity reached light-time levels at precisely the same time.

Acetyltransferases↗

Indoleamine metabolism in the pineal gland of the Chinese hamster, Cricetulus griseus.

Day- and nighttime contents of pineal melatonin and other indoleamines were measured in the Chinese hamster, Cricetulus griseus. The hamsters were inbred in our colony and kept in LD 14:10. Absolute levels of pineal melatonin and N-acetylserotonin contents were extremely low, but showed a significant circadian rhythm with a minor peak during the light period. Pineal hydroxytryptophan and serotonin (5HT) contents were also found to be higher in the light period. The activity of N-acetyltransferase in the pineal was higher in the light period than in the dark period, whereas there was no difference in hydroxyindole-O-methyltransferase activity between the light and dark periods. The uptake level of tryptophan into the pineal gland was higher in the light period than in the dark period. These results suggest that the 5HT synthesis pathway and/or tryptophan uptake may be involved in forming the circadian rhythm of 5HT content in the Chinese hamster pineal gland.

5-Hydroxytryptophan↗

Effects of cholinergic muscarinic agents on protein kinase C activity in rat pineal gland.

The role of protein kinase C (PKC) on muscarinic regulation of serotonin release in the pineal gland was investigated by measuring the pineal-PKC activity and serotonin secretion in response to muscarinic agents. Pineal slices, short-term incubated (0-15 min) without additions produced a low serotonin release and 20 to 24 percent PKC activity was found associated with membrane fractions. Prolonged exposure of pineal slices (30-180 min) produced further translocation of PKC activity to the membranes and a significant increase of serotonin release. Short-term treatment with pilocarpine and carbachol, stimulated PKC activity of both cytosolic and particulate fractions and the release of pineal serotonin. The pilocarpine effect was blocked by atropine indicating that it was mediated by muscarinic receptors. The present data support that PKC activation correlates with the increase of serotonin release by muscarinic agonist in pineal gland.

Angiotensin II↗

The weight of the pineal gland in malignancy.

A series of 150 pineal glands removed at routine postmortems in a general hospital have been examined. Statistical analysis of the weights of 147 of these glands from patients aged between 45 and 90 years, shows that the glands from patients dying of malignant disease are significantly lighter than those where the cause of death was non-malignant. These results are almost the exact reverse of those described recently in a similar series in America.After decalcification very little difference in the weight of the gland can be detected between the two groups and it would appear that the higher weight of the glands from non-malignant patients is due, at least in part, to the presence of a greater amount of mineral in these glands.

Aged↗

Audiogenic seizures and the pineal gland.

The effects of sound stimulation (electric bell, 110 dB) on the pineal glands of adult female rats were studied. Two types of animals were selected: audiogenic (Adg) and nonaudiogenic (Nadg). Unlike the Nadg rats, Adg rats exhibited tonic-clonic seizures in response to stimulation. In Adg rats, after a single seizure, all pinealocyte nuclei were pyknotic and the characteristic lobular organization of the pineal was markedly disrupted, indicating intense glandular stress; however, neither serotonin levels nor its biosynthesis were significantly altered. These results suggest a physiopathological relationship between audiogenic seizures and the pineal gland.

Acoustic Stimulation↗

The human pineal gland: a review of the "third eye" and the effect of light.

The human pineal gland is an extremely active neuroendocrine transducer. Environmental light acts through the retina and entrains the pineal gland's circadian rhythms by way of the hypothalamus and sympathetic nervous system. Light depresses pinealocyte activity. It is possible to do without the pineal, but this tiny gland is considered to be the "regulator of regulators" and important in general homeostasis. A direct retino-hypothalamic pathway is probably involved; and a system of synchronizing potentially independent oscillators is postulated.

Carbohydrate Metabolism↗

Neuropeptide Y-containing neurons in the rat superior cervical ganglion: projections to the pineal gland.

The aim of this study was to investigate the localization in the superior cervical ganglia (SCG) of neuropeptide Y-containing neurons innervating the pineal gland. Following injection of the fluorescent tracer Fluoro-Gold (FG) into the superficial part of the pineal gland and retrograde axonal transport, labeled cells were observed predominantly in the rostral third to half of SCG sections (average number 239 per ganglion). Incubation of the sections with neuropeptide Y (NPY) antiserum showed that the vast majority of neurons exhibit NPY-like immunoreactivity (NPY-LI). The comparison of cells labeled with FG and those containing NPY revealed that nearly three fourths of retrogradely labeled neurons also exhibit NPY-LI. Incubation of pineal gland sections with NPY antiserum showed immunoreactive axons, relatively sparse and scattered throughout the superficial part of the organ and the pineal stalk. The present results confirm the assumption that, in rodents, pineal NPY originates from the superior cervical ganglia.

Animals↗

Ultrastructural observations at pineal gland capillaries in four rodent species.

The fine structure of the capillaries of the pineal glands of the rat, mouse, chinchilla, and ground squirrel were investigated. The pineal endothelial cells in the rat, mouse and ground squirrel were often composed of attenuated cytoplasmic portions which contained numerous fenestrations, in contrast to pineal capillaries in the chinchilla which were lined by thick non-fenestrated endothelial cells. Marked morphological differences were also apparent in terms of the types of vesicles within the cytoplasm and abutting on the cell surface of pineal endothelial cells from the various species investigated. The interendothelial junctions exhibited remarkable species differences with the chinchilla pineal possessing typical tight endothelial junctions while those in the rat, mouse and ground squirrel lacked such endothelial cell associations. Generally, capillary lining cells in the chinchilla pineal resembled similar cells within the brain, while endothelial cells in pineal glands of rat, mouse and ground squirrel were more typical of those found in other endocrine organs. Species differences in the structure of the pineal capillaries may represent physiological differences as well.

Animals↗

Ultrastructure of the pineal gland in the adult dog.

The adult dog pineal gland was studied with the electron microscope. Pineal connective tissue spaces were poorly developed and showed capillaries with nonfenestrated endothelial cells. Two cell types, pinealocytes and astrocytes, could be identified in pineal parenchyma. Dog pinealocytes showed microtubules, centrioles, occasional cilia, and well-developed Golgi complexes. These cells showed thin processes with bulbous endings packed with vesicles. Astrocytes were characterized by the presence of numerous filaments. Their processes finished forming a glial layer bordering connective tissue spaces. The presence of myelinated and unmyelinated nerve fibers was also described.

Actin Cytoskeleton↗

Study of the post-natal effects of chemopreventive agents on ethylnitrosourea-induced transplacental carcinogenesis in rats. II. Influence of low-molecular-weight polypeptide factors from the thymus, pineal gland, bone marrow, anterior hypothalamus, brain cortex and brain white substance.

The influence of the polypeptide factors extracted from thymus, pineal gland, bone marrow, anterior hypothalamus, brain cortex or brain white substance on N-ethyl-N-nitrosourea (ENU)-induced transplacental carcinogenesis was studied in rats. ENU was given to pregnant rats as a single i.v. exposure at a dose of 75 mg/kg body weight on the 21st day of gestation. The polypeptide factors were given to the offspring as a series of s.c. injections, at a dose of 0.5 mg/rat/day, starting at one or 2.5 months of age and continuing throughout the whole of post-natal life. ENU induced tumors of the brain, spinal cord, peripheral nerves and kidneys in 94-98% of the offspring exposed to the carcinogen, with an average number of 2.3-2.6 tumors per rat, and an average survival time of 294 days. Post-natal thymus factor or pineal gland factor administration was followed by an increase in mean lifespan of approximately 2 months and a significant decrease (P < 0.05) in the total tumor number per tumor-bearing rat, as well as the incidence and multiplicity of spinal cord tumors. Pineal gland factor also decreased the incidence of peripheral nerve and kidney tumors and their number per tumor-bearing rat. Brain cortex factor and brain white substance factor treatment was followed by a decrease in total tumor multiplicity of 1.2- to 3.3-fold, and a decrease in incidence of brain tumors of 10 to 33% per rat in comparison to the controls. Brain cortex factor also decreased the total tumor incidence. At the same time, brain white substance factor administration increased the incidence of peripheral nerve tumors and decreased the mean lifespan. Both bone marrow factor and anterior hypothalamus factor did not have any modifying effects on any of the ENU-induced tumors and mean lifespan. Thus, our results show the possibility of attenuation of transplacental ENU-induced carcinogenesis with post-natal administration of some polypeptide substances.

Animals↗

Neuropeptide Y (NPY)-like immunoreactivity in peripheral and central nerve fibres of the golden hamster (Mesocricetus auratus) with special respect to pineal gland innervation.

Information on the ambient lighting conditions is conveyed from the retina to the pineal organ by a neuronal pathway involving the suprachiasmatic nucleus (SCN) which acts as a circadian pacemaker. In the hamster, circadian rhythms have been shown to be influenced by injection of neuropeptide Y (NPY) into the SCN. Since NPY-immunoreactive nerve fibres are present in the rat and guinea-pig pineal glands it appeared of interest to investigate the hamster pineal as part of the circadian rhythm generating/regulating system. For comparison kidney, small intestine and cerebral cortex were studied. Like in the other rodent species so far investigated only a few of the abundant sympathetic nerve fibres in the hamster pineal gland are NPY-immunoreactive, in contrast to the relatively rich innervation of the other organs. This speaks in favour of a possible central origin of pineal NPY-immunoreactive fibres. These may either exert vasoregulatory effects on pineal vasculature or be involved in the modulation of alpha-adrenergic receptor mediated regulation of pineal metabolism.

Animals↗

Selective inhibition of beta(2)-adrenergic receptor-mediated cAMP generation by activation of the P2Y(2) receptor in mouse pineal gland tumor cells.

Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)-coupled P2Y(2) receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol-stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC-coupled receptors including P2Y(1) and bombesin receptors had little or no effect on the isoproterenol-stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E(2), or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y(2) and beta(2)-adrenergic receptors. The binding of [(3)H]CGP12177 to beta(2)-adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol-stimulated cAMP production, whereas phorbol 12-myristate 13-acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca(2+), however, had little effect on the ATP-induced inhibition of cAMP production, while it completely reversed the ionomycin-induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide-induced increase in intracellular Ca(2+) and inositol 1,4,5-trisphosphate production by 30-40%, suggesting that the ATP-mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin-sensitive G(i)-protein. We conclude that one of the functions of P2Y(2) receptors on the pineal gland is the selective inhibition of beta-adrenergic receptor-mediated signaling pathways via the inhibitory G-proteins.

1-Methyl-3-isobutylxanthine↗

Effects of an artificial magnetic field on serotonin N-acetyltransferase activity and melatonin content of the rat pineal gland.

In the present study the effects of artificial magnetic fields on pineal serotonin-N-acetyltransferase (NAT) activity and melatonin content in male Sprague-Dawley rats were investigated to study the secretory activity of the pineal gland. Experimental inversion of the horizontal component of the natural magnetic field, performed at night-time, led to a significant decrease of both parameters investigated. During day-time, this effect was less conspicuous. During night-time, inversion of the horizontal component is followed by a reduced pineal secretory activity for about 2 h. After 24 h exposure to the inverted horizontal component, return to the natural condition was followed by a renewed clear depression of pineal NAT activity and melatonin content, indicating that the main stimulus is not the inverted magnetic field itself but rather its change. Changing the inclination of the local magnetic field from 63 degrees to 58 degrees, 68 degrees or 78 degrees, respectively also decreased the secretory activity of the rat pineal gland.

Acetyltransferases↗