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PubMed1994

Calmodulin, calbindin-D28K and calretinin in rat and chicken pineal glands: immunocytochemical and immunoblotting analysis.

In pineal gland, melatonin is synthesized in pinealocytes. Pharmacological studies using calmodulin antagonists suggested that melatonin synthesis was regulated through calmodulin. However, immunohistochemical studies showed that calmodulin could only be detected in pineal glial cells, and not in pinealocytes. To further investigate this discrepancy, we have tried to detect calmodulin not seen by immunohistochemical methods. We have used rat and chicken pineal homogenate supernatants and Triton X-100-treated pellets denatured by sodium dodecyl sulfate, subjected to electrophoresis and immunoblotting using anti-calmodulin antibodies. Two different IgG (#465 and #860) purified from anti-calmodulin sera were used. In rat pineal homogenate supernatants, calmodulin could be detected by immunoblotting using both antibodies. Some calmodulin could also be detected in the Triton-treated pellet fractions, but no additional cross-reacting bands were detected. However, in both chicken pineal homogenate supernatants and Triton-extracted pellets, in addition to a calmodulin immunoreactive band, two other proteins with approximate molecular masses (M(r)) of 56 kDa and 60 kDa were detected using anti-calmodulin #465. For comparison, similar immunoblot experiments were performed for detection of calbindin-D28K and calretinin, two other calcium binding proteins expressed in different pineal cell populations. Interestingly, Triton extraction of chicken pineal pellets revealed additional bands cross-reacting with each antibody. Anti-calbindin-D28K cross-reacted strongly with a M(r) = 68 kDa protein and weakly with a M(r) = 56 kDa protein. Anti-calretinin cross-reacted strongly with a M(r) = 93 kDa protein and weakly with a M(r) = 56 kDa protein.

Animals↗

PubMed1981

Embryonic development of the rat pineal gland.

The embryonic development of the albino rat pineal gland has been studied from day 13 of development until birth. The first pineal anlage appears as a midline evagination of the diencephalic roof, which soon adopts a tubular morphology. At 17 days, the disappearance of the pineal recess begins, along with the transformation of the gland into a solid organ. The latter is mainly achieved by an infolding and thickening of the dorsal recess wall, from which derives most of the future pineal parenchyma. Blood vessels are mainly derived from the vessels found in the dorsal surface of the pineal gland.

Animals↗

PubMed1992

Iodothyronine 5'-deiodinating activity in the pineal gland.

1. The presence of an iodothyronine 5'-deiodinating activity has been described in the pineal gland of various rodents, and it has been identified as a type II 5'-deiodinase isoenzyme since it is relatively insensitive to inhibition by propylthiouracil and its activity increases during hypothyroidism. 2. 5'-Deiodinase activity in the rat pineal gland follows a nyctohemeral profile, exhibiting basal values during the day and maximal values at night. The nocturnal increase is dependent on the noradrenergic input from the superior cervical ganglia, and both in vivo and in vitro studies show that beta-adrenergic receptors are primarily involved in the activation of the enzyme. 3. Day-night differences in rat pineal 5'-deiodinase activity are found beginning at 2 weeks of age, with rhythms increasing in amplitude until maximal differences are reached in adult animals. During the maturation of the rhythm, changes in regulation of enzyme activation are observed. Thus, during the first 2-3 weeks of age, alpha-adrenergic receptors appear to be as important as beta-adrenergic receptors in regulating the deiodinating activity of the pineal. However, in adults, no role of alpha-adrenergic receptors has been described. 4. Although regulation of 5'-deiodinase activity in the pineal gland is well established, few data are available concerning the physiological significance of the enzyme in the gland. Of the studies that have been performed, those attempting to demonstrate a relationship between pineal 5'-deiodinase activity and other pineal rhythms, e.g. those of melatonin production and N-acetyltransferase activity, indicates that the latter rhythms do not rely on the cyclic production of T3. The alternate possibility that the 5'D rhythm depends on the cyclic production of melatonin remains to be examined.

Animals↗

PubMed1988

Thyroxine 5'-deiodination in brown adipose tissue and pineal gland: implications for thermogenic regulation and role of melatonin.

T4 type II 5'-deiodinase (5'-D II) activity was studied in wild-captured Richardson's ground squirrels. As previously reported for other species, 5'-D II activity was detected in frontal cortex, cerebellum, pineal gland, and brown adipose tissue (BAT); in the median eminence the levels of 5'-D II activity were undetectable with our methodology. When pineal gland, frontal cortex, and cerebellum nyctohemeral profiles were studied, none of them showed variations. Cold exposure for 4 h led to an increase in the enzymatic activity 10-fold above the basal values for BAT, while in the pineal gland the values were doubled; cold exposure failed to change the 5'-D II activity in the frontal cortex. Acute melatonin treatment caused a 7-fold increase in 5'-D II activity in BAT, but did not affect enzyme activity in either the pineal gland or frontal cortex. The data indicate that 5'-D II in Richardson's ground squirrel shows classical localizations. Additionally, two new regulatory factors of 5'-D II are reported, i.e. melatonin for BAT and cold for the pineal gland.

Adipose Tissue, Brown↗

PubMedDate not supplied

Innervation of the rat pineal gland by nerve fibres originating in the sphenopalatine, otic and trigeminal ganglia. A retrograde in vivo neuronal tracing study.

The innervation of the rat pineal gland from the sphenopalatine, otic, superior cervical and trigeminal ganglia was investigated in animals by use of in vivo retrograde tracings. A solution of 2% Fluorogold was iontophoretically injected into the superficial pineal gland in a series of Wistar rats. After a survival time of 4-10 days, the animals were fixed by perfusion and the brains, sphenopalatine, otic, superior cervical and trigeminal ganglia were investigated with a fluorescence microscope. Many retrogradely labelled perikarya were found in the superior cervical ganglia, but a smaller number of neurones were also labelled in the sphenopalatine, otic and trigeminal ganglia. Injections of the tracer into the subarachnoidal space were used as the control for unspecific uptake and transport of the tracer. The input to the pineal gland from the parasympathetic sphenopalatine and otic ganglia might be involved in the regulation of the annual rhythms of the pineal gland. The projections from the sensory trigeminal ganglion could be involved in the control of the blood flow of the gland.

Afferent Pathways↗

PubMed1983

Depressive effect of LHRH on the numbers of "synaptic" ribbons and spherules in the pineal gland of diestrous rats.

Previous studies have shown that LHRH or LHRH-like substances are present in the pineal gland. In order to investigate whether exogenous LHRH may affect the pineal gland, in the present study the effects of a single dose of LHRH (1 microgram, i.p.) on pineal "synaptic" ribbons and spherules as well as serum melatonin levels were examined in diestrous Wistar rats. One hour after the injection both ribbons and spherules exhibited a statistically significant decrease in number. Serum melatonin levels were not affected. It is concluded that humoral feedback mechanisms may exist between the hypothalamus and the pineal gland.

Animals↗

PubMed2001

Central GABAergic innervation of the mammalian pineal gland: a light and electron microscopic immunocytochemical investigation in rodent and nonrodent species.

Light and electron microscopic immunocytochemical observations were made to demonstrate central pinealopetal fibers immunoreactive for gamma-aminobutyric acid (GABA) and synapses between their terminals and pinealocytes in the pineal gland of four rodent (Wistar-King rat; mouse; Syrian hamster, Mesocricetus auratus; Hartley strain guinea pig) and one nonrodent (tree shrew, Tupaia glis) species. GABA-immunoreactive myelinated and unmyelinated fibers and endings were found in the parenchyma of the pineal gland of all the animals examined. In the rodent species, GABAergic fibers were mainly found in the intermediate and proximal portions of the pineal gland and were nearly or entirely absent in the distal portion of the gland. Abundant GABAergic fibers were evenly distributed throughout the gland of the tree shrew. In all the animals, the habenular and posterior commissures contained abundant GABA-positive fibers, and some of them were followed to the pineal gland. GABA-positive endings made synaptic contact with pinealocytes, occasionally in mice and guinea pigs, and frequently in tree shrews; no synapses were observed in Syrian hamsters and rats. In the pineal gland of all the animals, GABA-immunoreactive cell bodies were not detected, and sympathetic fibers were not immunoreactive for GABA. These data indicate that GABAergic fibers are main pinealopetal projections from the brain. In view of the difference in the distribution of these fibers, central GABAergic innervation may play a more significant role in nonrodents than in rodents. The frequent occurrence of GABAergic synapses on pinealocytes in the tree shrew suggests that GABA released at these synapses directly controls activity of pinealocytes of this animal.

Animals↗

PubMed1994

Melatonin deacetylase activity in the pineal gland and brain of the lizards Anolis carolinensis and Sceloporus jarrovi.

Melatonin modulates a variety of rhythmic processes in vertebrates, and is synthesized in both the retina and pineal gland. We have shown previously that retinal melatonin is deacetylated generating 5-methoxytryptamine, which is then deaminated by monoamine oxidase, producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. This process occurs within the eyes of a variety of vertebrates including the iguanid lizard Anolis carolinensis. To determine whether melatonin deacetylase activity also occurs in the pineal organ or in other parts of the lizard brain, pineals and brains of Anolis carolinensis and Sceloporus jarrovi were cultured in the presence of [3H-methoxy]-melatonin. High-performance liquid chromatography of the resulting culture media and tissues revealed the generation of radiolabeled 5-methoxytryptamine and 5-methoxyindoleacetic acid. These two methoxyindoles were the only radiolabeled metabolites detectable, and together accounted for all melatonin lost. Both the loss of melatonin and the production of melatonin metabolites were inhibited by inclusion of 100 microM eserine, an inhibitor of the melatonin deacetylase. Pargyline, a monoamine oxidase inhibitor, reduced the production of 5-methoxyindoleacetic acid and increased the production of 5-methoxytryptamine relative to control incubations. Similar effects of eserine and pargyline were seen in eyecup, brain and pineal gland, but the specific activity of melatonin deacetylation in cultured pineal glands was much greater than in either brains or eyecups. These results indicate that pineal glands of both Anolis carolinensis and Sceloporus jarrovi can rapidly catabolize melatonin by a mechanism very similar to that in the eye, that the melatonin deacetylation pathway exists elsewhere in the iguanid brain, and also extend our previous observations of ocular melatonin deacetylation to an additional species.

5-Methoxytryptamine↗

PubMed1998

Neuroimmunomodulation of aging. A program in the pineal gland.

We have investigated for 35 years the relationship between the neuroendocrine and the thymo-lymphatic, immune system. In the last decade we have shown that the pineal gland is a main adapter and fine synchronizer of environmental variables and endogenous messages into physiological modifications of basic functions. In particular the pineal gland itself seems to regulate, via circadian, night secretion of melatonin, all basic hormonal functions and also immunity. We have shown with several in vivo models that this fundamental role of the pineal gland decays during aging. Aging itself seems to be a strictly pineal-programmed event similar to growth and puberty. The continuation of our interventions with melatonin against the typical degenerative diseases of aging must be based on an accurate evaluation of its mechanisms of action. Melatonin being a ubiquitous molecule in nature, we suggest that it has acquired during evolution of the species numerous levels of activities. In fact, melatonin can be found in a large variety of cells and tissues, and bindings sites and "receptors" have been identified in many tissues and cells of the neuroendocrine and immune system. Therefore, the progressive understanding of the aging-programming role of the pineal gland also depends on studies of melatonin and its basic regulatory function. Our present studies will be described.

Aging↗

PubMed2006

LIM homeodomain proteins Islet-1 and Lim-3 expressions in the developing pineal gland of chick embryo by immunohistochemistry.

LIM homeodomain proteins Islet-1 and Lim-3 expression and their role in nervous tissue and endocrine glands have been reported; however, nothing is known concerning Islet-1 and Lim-3 expression in the developing pineal gland of the chick embryo. The aim of the present study was to determine the ontogeny of Islet-1 and Lim-3 expression in the developing pineal gland of chick embryo using immunohistochemistry. The results showed that Islet-1 and Lim-3 immunopositive cells were first detected in the pineal evagination of chick embryos at day 4 (E4) and E4.5 of incubation, respectively. In the later developing stages, both Islet-1 and Lim-3 immunopositive cells were consistently detected in the follicular and parafollicular pinealocytes throughout the pineal gland. The relative percentage of Islet-1 immunopositive (Islet-1+) cells relative to the total cells was about 6% at E4.5, and then kept increasing (P < 0.05) and reached about 40% by E12.5; this was followed by no obvious changes until the chicks were newly hatched. The change in Lim-3 immunopositive (Lim-3+) cell number was parallel to that of Islet-1, although Lim-3+ cell were significantly fewer than Islet-1+ cell numbers from E4.5 to E8.5 (P < 0.05). Dual immunohistochemical staining results showed that almost all the Lim-3+ cells expressed Islet-1 at every stage examined, and about 90% of Islet-1+ cells were proliferating cell nuclear antigen negative. These results suggest that both Islet-1 and Lim-3 may be involved in regulating the development and functional maturation of the pineal gland, although further studies are required in elucidating the functional roles of Islet-1 and Lim-3 and the related mechanisms.

Animals↗

PubMed2005

UV-A light regulation of arylalkylamine N-acetyltransferase activity in the chick pineal gland: role of cAMP and proteasomal proteolysis.

Acute exposure of dark-adapted, cultured chick pineal glands to UV-A light significantly decreased the tissue cAMP concentration and the activity of arylalkylamine N-acetyltransferase (AANAT), the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway. The magnitude of these changes was dependent on the duration of UV-A exposure. The UV-A light-evoked decline in pineal AANAT activity was blocked by cAMP protagonists (forskolin and dibutyryl-cAMP) and by inhibitors of the proteasomal degradation pathway (MG-132, proteasome inhibitor I, and lactacystin). These results indicate that the chick pineal gland is directly sensitive to UV-A light. By analogy to white light, the suppressive action of UV-A radiation on AANAT activity in the chick pineal gland involves changes in the tissue cAMP level and enhanced proteasomal proteolysis.

Acetylcysteine↗

PubMed1990

Autoradiographic assessment of pineal gland glucose utilization and capillary permeability in the unanesthetized rat.

Pineal gland glucose utilization (GU) and capillary permeability (CP) were measured in unanesthetized rats, using complementary quantitative autoradiographic techniques. GU values within the pineal tissue were homogeneously distributed around 70 mumol of glucose/100 g each min, i.e., they were approximately 30% lower than in the cortical gray structures. The blood-to-brain transfer constant of [14C]-alpha-aminoisobutyric acid, as an index of CP, was up to ten orders of magnitude higher than that for the rest of the brain. These measurements were carried out at that point in the circadian rhythm that corresponds to the minimum level of neurosecretory activity of the pineal gland.

Aminoisobutyric Acids↗

PubMed2000

Direct suppressive effects of weak magnetic fields (50 Hz and 16 2/3 Hz) on melatonin synthesis in the pineal gland of Djungarian hamsters (Phodopus sungorus).

In many investigations performed thus far on rodents, a suppression of melatonin synthesis was observed when animals were exposed to weak magnetic fields. However, among the several issues not yet resolved is the question of whether the observed changes are caused by direct effects on the pineal gland or by indirect effects, e.g., at the level of the eyes. We, therefore, performed a series of experiments in which direct effects of weak magnetic fields were studied in isolated pineal glands of Djungarian hamsters (Phodopus sungorus). After sacrifice of animals during morning hours, pineal glands were removed and placed individually into glass chambers that were perfused with oxygenated buffer. Experiments (n = 8) lasted for 8 hr. Magnetic fields (1,623 or 50 Hz at 86 microTesla) were generated by Helmholtz coils, and were present during the entire period. In each experiment, 12 exposed, and 12 sham-exposed, pineal glands were placed individually in glass chambers, which were surrounded by identical coils. Because of the design of the coils (bifilament), and the experimental setup (black box), the experimentator was not aware which coil was activated until the results were obtained. After 3.25 hr of adaptation, melatonin production was stimulated by isoproterenol (10(-7) M) for 30 min. Eluted fractions were collected, and melatonin concentrations were measured by radioimmunoassay. Maximum melatonin production was achieved after 5-7 hr. In all experiments, maximum melatonin concentrations were lower in the exposed groups compared with the sham-exposed controls. Statistical analyses for each frequency showed significant suppressive effects at 16(2/3) Hz (P < 0.01), and 50 Hz (P < 0.00). It is concluded that the suppressive effects of magnetic fields on the synthesis of melatonin are a result of primary mechanisms at the level of the pineal gland.

Animals↗

PubMed1979

Electrophysiology and pharmacology of striated muscle fibres cultured from dissociated neonatal rat pineal glands.

1. Striated muscle fibres were found in each of twenty consecutive pineal glands cultured from individual neonatal rats.2. In subsequent experiments performed with dissociated cultures of pineal organs pooled from several litters, myotubes were first visible after about 1 week in culture.3. During the next several weeks the myotubes increased in size, developed crossstriations, and began to twitch spontaneously.4. The resting membrane potential increased with age in culture. All myotubes studied showed delayed rectification. Action potentials either occurred spontaneously or could be evoked if the membrane were sufficiently polarized. No spontaneous end plate potentials were seen.5. Acetylcholine (ACh) produced a brief, monophasic depolarizing response. Noradrenaline, serotonin, melatonin, dopamine, and gamma-aminobutyric acid (GABA) had no effect on the resting membrane potential when applied iontophoretically.6. The ACh response was reversibly blocked by 10(-6)M-tubocurarine and irreversibly blocked by 10(-6)M-alpha-bungarotoxin. Atropine (10(-4)M) reduced the amplitude and shortened the time course of the ACh response, and 10(-3)M-atropine produced complete but reversible inhibition.7. We conclude that pineal muscle fibres are electrophysiologically and pharmacologically similar to skeletal muscle fibres in vitro. Although the pineal gland has undetectable levels of ACh, pineal muscle develops ACh receptors but not noradrenaline, serotonin, melatonin, dopamine, or GABA receptors mediating electrophysiological responses, although these latter substances (except dopamine) are found in the pineal.

Acetylcholine↗

PubMed1988

Identification of alpha 2-adrenergic receptors in chicken pineal gland using [3H]rauwolscine.

The norepinephrine-induced inhibition of avian pineal N-acetyltransferase activity appears to be mediated by alpha 2-adrenergic receptors. In this study, alpha 2-adrenergic receptors in the chicken pineal gland were directly identified by radioligand binding. Membrane preparations of pineal glands from chickens from 1 to 6 weeks of age were examined using [3H]rauwolscine, a selective alpha 2-adrenergic receptor antagonist, to characterize the binding sites. The results indicate no ontological change in either the affinity (KD) or density of receptor binding sites (Bmax) during the time span examined. The binding was saturable and of high affinity with a mean KD of 0.27 +/- 0.01 nM and a mean Bmax of 242 +/- 12 fmol/mg protein. Further characterization of these binding sites indicated that the alpha 2-adrenergic receptor is of the alpha 2A subtype, since prazosin and ARC-239 bound with low affinities and oxymetazoline bound with high affinity.

Adrenergic alpha-Antagonists↗

PubMed2006

Photoperiodism as a modifier of effect of extremely low-frequency electromagnetic field on morphological properties of pineal gland.

The aim of our study was to determine, using histological and stereological methods, whether photoperiodism has any impact on the effects that chronic (three-month long) exposure to LF-EMF (50Hz) has on morphological characteristics on rat's pineal gland. The experiment was performed on 48 Mill Hill male rats (24 experimental and 24 control). Upon birth, 24 rats were exposed for 7h a day, 5 days a week for 3 months to LF-EMF (50 Hz, 50-500microT, 10V/m). In the winter (short days, long nights), the activity of the pineal gland and neuroendocrine sensitivity is increased. The study was performed both during summer and winter, following the identical protocol. After sacrifice of animals, samples of pineal gland were processed for HE staining and then were analyzed using the methods of stereology. The most significant changes in epiphysis in the first group of animals in wintertime are: altered glandular feature, hyperemia, reduced pinealocytes with pale pink, poor cytoplasm and irregular, stick-form nuclei. In the second group (II) pinealocytes are enlarged, with vacuolated cytoplasm and hyper chromatic, enlarged nucleus. Morphological changes of pineal gland at rats in the summertime were not as intense as in the winter and finding of the gland in the group II is compatible with those from the control group. Stereological results show both in winter and summer in the first group the decrease of volume density of pinealocytes, their cytoplasm and nuclei and in the second group in winter increase the volume density of pinealocytes, cytoplasm and nuclei, while in the second group the results in summertime are equal to those from the control group. Photoperiodism is modifier of effect of LF-EMF on morphological structure of pineal gland, because the gland recovery is incomplete in winter and reversible in summer.

Animals↗

PubMed2000

Transcription factor dynamics and neuroendocrine signalling in the mouse pineal gland: a comparative analysis of melatonin-deficient C57BL mice and melatonin-proficient C3H mice.

In rodents, the nocturnal rise and fall of arylalkylamine N-acetyltransferase (AANAT) activity controls the rhythmic synthesis of melatonin, the hormone of the pineal gland. This rhythm involves the transcriptional regulation of the AANAT by two norepinephrine (NE)-inducible transcription factors, e.g. the activator pCREB (phosphorylated Ca2+/cAMP-response element binding protein) and the inhibitor ICER (inducible cAMP early repressor). Most inbred mouse strains do not produce melatonin under standard laboratory light/dark conditions. As melatonin-deficient mice are often the founders for transgenic animals used for chronobiological experimentations, molecular components of neuroendocrine signalling in the pineal gland as an integral part of clock entrainment mechanisms have to be deciphered. We therefore compared calcium signalling, transcriptional events and melatonin synthesis in the melatonin-deficient C57BL mouse and the melatonin-proficient C3H mouse. Pineal glands and primary pinealocytes were cultured and stimulated with NE or were collected at various times of the light/dark (LD) cycle. Changes in intracellular calcium concentrations, the phosphorylation of CREB, and ICER protein levels follow similar dynamics in the pineal glands of both mouse strains. pCREB levels are high during the early night and ICER protein shows elevated levels during the late night. In the C57BL pineal gland, a low but significant increase in melatonin synthesis could be observed upon NE stimulation, and, notably, also when animals were exposed to long nights. We conclude that the commonly used C57BL mouse is not completely melatonin-deficient and that this melatonin-deficiency does not affect molecular details involved in regulating transcriptional events of melatonin synthesis.

Adrenergic alpha-Agonists↗

PubMed1979

Histochemical demonstration of a circadian rhythm of succinate dehydrogenase in rat pineal gland. Influence of coenzyme Q10 addition.

Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The influence of fixation on the activity pattern, the possible diffusion of enzyme, the nothing dehydrogenase reaction, and the substantivity of the tetrazolium salts and formazans were investigated in control experiments. In rats maintained on a 17/7 h light/dark schedule a distinct circadian rhythm of the succinate dehydrogenase was demonstrated in the pineal gland. Activity was lowest during the day and highest during the night. The dorsocaudal part of the gland showed the highest activity and within the same part of the gland the activity varied between individual pinealocytes. A relative lack of endogenous coenzyme Q, as well as a circadian rhythm of this coenzyme, highly influenced the activity of succinate dehydrogenase. It is concluded that succinate dehydrogenase activity in the pineal gland of the rat is regulated by changing the concentration of the active enzyme itself as well as the level of the endogenous coenzyme Q. Whether this is caused by a circadian rhythm in the synthesis or in the catabolism of the enzyme and the coenzyme was not revealed by the present study .

Animals↗