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The pineal gland and mammalian photoperiodism.

The mammalian pineal gland appears to be a major endocrine component in the regulation of photoperiodic responses. The circadian pattern of secretion of the pineal hormone, melatonin, is regulated by the nervous system. Changes in photoperiod, acting via the nervous system, alter the temporal pattern of melatonin secretion. The changes in secretion pattern convey information about daylength from neural components of the circadian system to the reproductive system, and probably to other physiological systems.

Animals↗

Immunohistochemical evidence for the presence of melatonin in the pineal gland, the retina and the Harderian gland.

The presence of melatonin is demonstrated in the pineal gland, the retina and the Harderian gland in some mammalian and non-mammalian vertebrates, using a specific fluorescence labelled antibody technique. Four different potent antibodies against melatonin have been used and compared. In the pineal gland of hamsters, mice, rats and snakes, specific fluorescence, mostly restricted to the cytoplasm of the cells, is detected in pinealocytes. Fluorescence is also detected in the pineal organ of fishes, tortoises and lizards, but it has not been possible, from cryostat sections of fresh tissue, to assert which kind of cell is reacting (photoreceptor cells or interstitial ependymal cells). In the retina, fluorescence is almost exclusively restricted to the outer nuclear layer. In the Harderian gland of mammals and reptiles, fluorescence is localized in the secretory cells of the alveoli and mostly restricted to the cytoplasm surrounding the nucleus. These results are discussed in relation to the concept of melatonin synthesis at extrapineal sites independent of pineal production.

Animals↗

Reciprocal interdependence between pineal gland and avian immune system. NEL Review.

The immune system of mammals and birds exhibits the same basic anatomical and functional organization, including dichotomy into the cellular and humoral immune response. Specificities of avian immune system may be, however, very useful for understanding numerous phylogenetic and evolutionary mysteries. Similarities and differences between mammals and birds in terms of several pineal gland functions are well known, and they seem to include the immunomodulatory activity of melatonin (MEL) as well. Embryonic pinealectomy of the chicken demonstrated functional interrelationships between the development of the pineal gland, immune system and/or neuroendocrine network, and embryonic bursectomy influenced the diurnal rhythm of the pineal gland function and abolished the effect of immunization on serum MEL level. Also immunization with a thymo-dependent antigen (SRBC) evoked some changes in the chicken nocturnal pineal NAT activity. We have found that the pineal gland and MEL control the diurnal rhythm of immunity in the chicken, but we were not able to demonstrate any immunostimulatory and anti-glucocorticoid MEL effects, regardless of the chicken's age, sex, season, and hormone dose used. The existence of functional connections between the pineal gland and the immune system in chickens was, however, confirmed in other experimental approaches. Specific and reversible binding of 2-[125I]iodoMEL to the membrane preparations from lymphoid glands was demonstrated in several avian species. In vitro MEL diminished lymphocyte proliferation stimulated by the common T-cell mitogens, while alone failed to influence the blast formation. Reciprocal functional connections between the avian immune system and the pineal gland seem to be well documented, but the mechanism(s) involved have to be elucidated.

Journal Article↗

Circadian cycles in VIP content and VIP stimulation of cyclic AMP accumulation in the rat pineal gland.

VIP content in the rat pineal gland and cyclic AMP accumulation in response to VIP in the daily light and dark cycle were examined. VIP content in the pineal varied significantly during the day and night; the content decreased during exposure to light and was lowest at the onset of darkness, 6 p.m. (mean +/- SE, 23 +/- 5 pg/pineal), and increased during the night and was highest at the onset of light, 6 a.m. (72 +/- 12 pg/pineal). Response of cyclic AMP accumulation to VIP varied with a periodicity inversely related to the daily light and dark cycle of VIP content; cyclic AMP accumulation in response to 10(-7) M VIP increased in proportion to periods of exposure to light and peaked at 6 p.m., and decreased with the onset of darkness.

Animals↗

Melatonin synthesis in the bovine pineal gland is regulated by type II cyclic AMP-dependent protein kinase.

We investigated the expression of regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase (cAK; ATP:protein phosphotransferase; EC 2.7.1.37) in the bovine pineal gland. In total RNA extracts of bovine pineal glands moderate levels of RI alpha/RII beta and high levels of C alpha and C beta mRNA were found. We were able to detect a strong signal for RII and C subunit at the protein level, whereas RI was apparently absent. Probing sections of the intact bovine pineal gland with RI and RII antibodies stained only RII in pinealocytes. Pairs of cyclic AMP analogues complementing each other in activation of type II cAK, but not cAKI-directed analogue pairs, showed synergistic stimulation of melatonin synthesis. Moreover, melatonin synthesis stimulated by the physiological activator norepinephrine in pineal cell cultures was inhibited by cAK antagonists. Taken together these results show the presence of RII regulatory and both C alpha and C beta catalytic subunits and thus cAKII holoenzyme in the bovine pineal gland. The almost complete inhibition of norepinephrine-mediated melatonin synthesis by the cAK antagonists emphasizes the dominant role of cyclic AMP as the second messenger and cAK as the transducer in bovine pineal signal transduction.

Animals↗

Inhibition of avian and mammalian hydroxy-indole-o-methyl-transferase (HIOMT) with low molecular weight fractions of mammalian pineal glands.

Partially purified fractions from sheep pineal gland extracts, which show a fluorescence like pteridines, were shown to suppress the enzyme hydroxy-indole-o-methyltransferase (HIOMT) isolated from avian pineals and retinas. The results described demonstrate that the mechanism of inhibition of HIOMT activity is independent of the species and organ. A blocking effect was found in experiments whether retinal or pineal HIOMT were used and the effect was found as well with bovine HIOMT. It is shown that low molecular weight molecules show inhibitory activity and accompany the high molecular weight enzyme HIOMT.

Acetylserotonin O-Methyltransferase↗

Changes in vasopressin-converting aminopeptidase activity in the rat pineal gland during summer: relationship to vasopressin contents.

Vasopressin (VP)-converting aminopeptidase (VP-AP) activity and VP contents were measured in single rat pineal glands during the summer of two successive years. The peptidase activity decreased significantly in August. The lowest activity (+/- SEM) of 0.18 +/- 0.02 pmol.hour-1 was recorded on August 14, compared to the basal activity of 0.25 +/- 0.01 pmol.hour-1 in July and September of 1986. The change with similar percentage occurred in the same period of 1987. The specific activity of the enzyme in the crude homogenate, 15,000 g pellet and supernatant fraction of rat pineal glands, exhibited the same pattern of variations. The decrease in peptidase activity coincided with the previously reported dramatic rise in pineal VP content in early August which was confirmed in this series of experiments. Another peptidase, the so-called gamma-endorphin generating endopeptidase (gamma-EGE) activity, and beta-endorphin-related peptides in the pineal gland did not change in this period. The results show that the variations of pineal VP contents and VP-AP activity during summer are not general for other peptides and peptidases. The coincidence of opposite changes in VP content and VP-AP activity of the pineal gland may indicate a role of the peptidase activity to regulate the VP content.

Acclimatization↗

Symptomatic glial cysts of the pineal gland.

Small asymptomatic cysts of the pineal gland represent a common incidental finding in adults undergoing computerized tomography or magnetic resonance (MR) imaging or at postmortem examination. In contrast, large symptomatic pineal cysts are rare, being limited to individual case reports or small series. The authors have reviewed 24 cases of large pineal cysts. The mean patient age at presentation was 28.7 years (range 15 to 46 years); 18 were female and six male. Presenting features in 20 symptomatic cases included: headache in 19; nausea and/or vomiting in seven; papilledema in five; visual disturbances in five (diplopia in three, "blurred vision" in two, and unilateral partial oculomotor nerve palsy in one); Parinaud's syndrome in two; hemiparesis in one; hemisensory aberration in one; and seizures in one. Four lesions were discovered incidentally. Magnetic resonance imaging typically demonstrated a 0.8- to 3.0-cm diameter mass (mean 1.7 cm) with homogeneous decreased signal intensity on T1-weighted images, increased signal intensity on T2-weighted images, and a distinct margin. Hydrocephalus was present in eight cases. The cysts were surgically excised via an infratentorial/supracerebellar approach (23 cases) or stereotactically biopsied (one case). Histological examination revealed a cyst wall 0.5 to 2.0 mm thick comprised of three layers: an outer fibrous layer, a middle layer of pineal parenchymal cells with variable calcification, and an inner layer of hypocellular glial tissue often exhibiting Rosenthal fibers and/or granular bodies. Evidence of prior hemorrhage, mild astrocytic degenerative atypia, and disorganization of pineal parenchyma were often present. Postoperative follow-up review in all 24 cases (range 3 months to 10 years) revealed no complications in 21, mild ocular movement deficit in one, gradually resolving Parinaud's syndrome in one, and radiographic evidence of a postoperative venous infarct of the superior cerebellum with ataxia of 1 week's duration in one. Of the patients referred for study, the cysts were most often initially misdiagnosed as a pineocytoma in eight and a pilocytic astrocytoma in three. Only two patients were correctly diagnosed as having pineal cysts. This stresses the importance of recognizing the histopathological spectrum of pineal cysts, as well as correlation with radiographic findings, if a correct diagnosis is to be attained.

Adolescent↗

Expression of the gene for the retinal protein peripherin in the pineal gland of humans and Djungarian hamsters (Phodopus sungorus).

This study investigated whether the gene for peripherin, a protein previously considered to play only a role in the retina, may also be expressed in the pineal gland of both Djungarian hamsters (Phodopus sungorus) and humans. After extraction of mRNA from pooled pineal glands of hamsters and from one human pineal gland, mRNA was transcribed into cDNA followed by PCR amplification with specific primers. Clear signals were obtained at the expected sizes of the PCR products. An additional experiment in hamsters revealed that the peripherin gene is expressed throughout the entire 24-h period. Since peripherin is an important protein in the retina stabilizing the photoreceptor discs it is speculated that this protein might also play a structural role in the pineal gland.

Animals↗

Developmental expression of tryptophan hydroxylase mRNAs in the rat pineal gland: an in situ hybridization study.

The expression of messenger RNAs encoding for tryptophan hydroxylase (TPOH), the first enzyme involved in serotonin and melatonin synthesis, has been investigated by in situ hybridization during the development of the rat pineal gland. TPOH mRNAs were detected as early as the twentieth day of gestation (E20) in the rat embryo before any nerve ending was observed in the pineal gland. After birth, their expression increased strongly, and attained a plateau during the second week. This coincides with the setting up of sympathetic innervation. From day 17 (D17), the TPOH mRNA expression diminished. These results indicate that noradrenergic innervation is not involved in the initiation of rat pinealocyte differentiation, but might modulate cell maturation. This study showed the existence of three types of cells arranged in patches in the young rat pineal gland (D6): regions in which cells expressed TPOH mRNAs, regions in which cells expressed vimentin, an intermediate filament protein present in the cytoskeleton of immature cells, and regions in which both TPOH mRNAs and vimentin are expressed. In older rat pineal gland (D20), almost all cells express TPOH mRNAs, and some cells still express vimentin. This suggests that all cells do not reach the same level of differentiation at the same time in the rat pineal gland.

Animals↗

Norepinephrine stimulates testosterone aromatization and inhibits 5 alpha reduction via beta-adrenoceptors in rat pineal gland.

The possible modulatory role of the sympathetic nervous system on testosterone aromatization and 5 alpha reduction by rat pineal gland was examined in vitro. NE (10 microM) added to pineal organ cultures increased by 72% the conversion of [14C]testosterone into estradiol and depressed by 39 and 53% that into the 5 alpha-reduced metabolites 5 alpha-dihydrotestosterone (5-DHT) and 5 alpha-androstanediol (5 alpha-diol). Both effects of NE were negated by the addition of the beta-adrenoceptor antagonist propranolol but not by the alpha-adrenoceptor antagonist phentolamine. Dibutyryl cAMP (0.1 mM) mimicked the effect of NE on pineal [14C]testosterone metabolism; it also mimicked the NE-induced inhibition of [14C]progesterone reduction to 5 alpha-pregnanedione and 3 alpha-hydroxy-5 alpha-pregnan-20-one by rat pineal gland explants. At the end of the dark phase of the daily photoperiod, pineal aromatization of testosterone was significantly higher, and 5 alpha reduction lower, than in rats killed at noon. Pineal glands obtained from rats subjected to superior cervical ganglionectomy 12 h earlier exhibited increased conversion of [14C]testosterone into estradiol, and depressed synthesis of 5 alpha-reduced metabolites, as compared with their respective sham-operated controls. 3 days after ganglionectomy a diminished testosterone aromatization was found. These results suggest that the increased release of NE from pineal sympathetic nerve endings stimulates testosterone aromatization and inhibits 5 alpha reduction via a beta-adrenoceptor.

5-alpha Reductase Inhibitors↗

Microdialysis of melatonin in the rat pineal gland: methodology and pharmacological applications.

The present study describes the development of a new technique to measure melatonin contents in the pineal gland of freely moving rats, by means of on-line microdialysis. The transcerebral cannula was modified, and a sensitive assay of melatonin, using HPLC with fluorimetric detection, was set up. With this system it is possible to monitor the melatonin levels on-line in the pineal gland during day- and nighttime. The nightly increase in melatonin release was recorded. Tetrodotoxin had an inhibitory effect on nighttime levels, whereas even high concentrations did not alter the daytime level. From this we conclude that neuronal activity is necessary to synthesize melatonin and that during daytime no net neuronal activity is present. Melatonin levels could be greatly enhanced by systemic administration of the beta-agonist isoprenaline (ISO). Also, local infusion of ISO or 8-bromoadenosine 3',5'-cyclic monophosphate, an analogue of the second messenger cyclic AMP, resulted in increased melatonin levels, demonstrating the presence of beta-adrenergic receptors, coupled to a cyclic AMP-based second messenger system, on the pineal gland. Injection of phenylephrine had no effect on daytime levels. Only when administered during ISO-induced stimulation of melatonin release did it enhance this stimulated release. This proved the regulatory role of alpha 1-receptors on pinealocytes. The method presented is of special interest for investigating the innervation of the pineal gland and the biochemical processes that regulate the biosynthesis of melatonin. Also, for studies on the diurnal rhythms of melatonin release and factors that influence these rhythms in freely moving animals, this model will be of great value.

8-Bromo Cyclic Adenosine Monophosphate↗

Possible involvement of neuropeptide Y in the seasonal control of hydroxyindole-O-methyltransferase activity in the pineal gland of the european hamster (Cricetus cricetus).

Hydroxyindole-O-methyltransferase (HIOMT) catalyses the last step of all the 5-methoxyindoles synthesized in the pineal gland. The synthetic activity of this neuroendocrine structure is driven not only by noradrenaline but also by various neuropeptides. Recently we have established (1) that one of these neuropeptides, neuropeptide Y (NPY), stimulates specifically HIOMT activity in rat pinealocytes and (2) that the density of the NPY-immunoreactive (NPY-IR) fibers innervating the pineal gland of the European hamster (Cricetus cricetus) displays seasonal variations with a large increase in the late autumn. These findings have led us to evaluate a possible seasonal control of NPY on the European hamster pineal gland. We thus compared the nycthemeral patterns of pineal HIOMT activity and 5-methoxytryptophol (5-ML) content and of circulating MEL levels in European hamsters when NPYergic innervation is low (end of October) and when it is the highest (mid-December). We report in this study that HIOMT activity is significantly increased by 80% in mid-December compared with end of October. This increase is correlated with the appearance of a nycthemeral rhythm of pineal 5-ML levels (with a fourfold increase occurring in early dawn and decreasing slowly towards the end of the day). These observations suggest that NPY could be an important neurotransmitter involved in the seasonal control of the biochemistry of the European hamster pineal gland via a stimulatory effect on HIOMT activity.

Acetylserotonin O-Methyltransferase↗

Action spectrum of the retinal mechanism mediating nocturnal light-induced suppression of rat pineal gland N-acetyltransferase.

The spectral properties of the retinal mechanism mediating the inhibitory effects of nocturnal light on pineal gland N-acetyltransferase (NAT) activity were determined. Pineal gland NAT activity declined linearly in albino rats exposed to different irradiances of a 460 or 580 nm monochromatic light during the middle of the dark phase of the cycle. The difference in sensitivity to the test lights is that predicted for a photopigment having peak absorbance at 495 nm, suggesting the inhibition of pineal gland N-acetyltransferase activity is mediated by the photopigment found in rat rods.

Acetyltransferases↗

Tyrosine hydroxylase activity in peripherally denervated rat pineal gland.

The presence of tyrosine hydroxylase (TH) in the rat pineal gland was studied using a combination of immunochemical and biochemical methods. In superior cervical ganglionectomized (SCGx) animals and in isolated pineals incubated for 72 h, both TH immunoreactive (TH-IR) fibers and TH biochemical activity were still present but reduced. Conversely, in dispersed pinealocytes incubated for only 24 h we were unable to detect either TH activity or TH-positive cells. Since in the pineal gland of intact rats total 3-methoxy-4-hydroxy phenylglycol (MHPG) was undetectable, and only traces of norepinephrine (NE) were present in the pineal of ganglionectomized animals, the results suggest a central pinealopetal catecholaminergic pathway which could use dopamine as a neurotransmitter.

Animals↗

Molecular cloning and nucleotide sequence of a cDNA encoding hydroxyindole O-methyltransferase from chicken pineal gland.

Hydroxyindole O-methyltransferase (EC 2.1.1.4) is the enzyme that catalyses the synthesis of melatonin in the pineal gland and in the retina. Polyadenylated RNA from chicken pineal glands was used to prepare a cDNA library in lambda gt11. The library was screened with an antiserum directed against chicken hydroxyindole O-methyltransferase, and one cDNA clone was isolated. The fusion protein expressed by phage lysogens was identified on Western blots as a 165 kDA immunoreactive protein (beta-galactosidase, 110 kDa; hydroxyindole O-methyltransferase, 38 kDa). The fusion protein exhibited hydroxyindole O-methyltransferase activity. Its Km values for N-acetyl-5-hydroxytryptamine and S-adenosylmethionine were 5 times those of the natural enzyme. The intrinsic activity of the fusion protein was approx. 0.25% that of the natural enzyme. The cDNA consisted of 1436 nucleotides, including a 1038-nucleotide sequence encoding a full-length 346-amino-acid hydroxyindole O-methyltransferase. Comparison with bovine hydroxyindole O-methyltransferase [Ishida, Obinata & Deguchi (1987) J. Biol. Chem. 262, 2895-2899] revealed 52% identity in nucleotide sequences and 44% identity in peptide sequences. Northern-blot analysis revealed the presence of hydroxyindole O-methyltransferase mRNA transcripts in chicken pineal gland and retina, but not in the telencephalon.

Acetylserotonin O-Methyltransferase↗

Interaction of angiotensin II with the cholinergic and noradrenergic systems in the rat pineal gland: regulation of indole metabolism.

The pineal gland, angiotensin, and noradrenergic and cholinergic systems are involved in the regulation of tissue indole metabolism. Angiotensin II increased noradrenaline release and the production of hydroxy- and methoxyindoles by pineal slices. Electrical field stimulation (EFS) of pineal slices released angiotensin II and reproduced many of the actions of exogenous angiotensin II on serotonin and melatonin biosynthesis and release. Both sarcosine-isoleucine-angiotensin II ([Sar, Ile]-ANG II) and atropine blocked, and nadolol increased, the effect of EFS and exogenous angiotensin II on serotonin production. Nadolol blocked both the EFS-induced and the angiotensin II-induced production of melatonin. Atropine and [Sar, Ile]-ANG II did not modify melatonin biosynthesis in electrically stimulated slices, but the muscarinic receptor antagonist increased the stimulatory effect of angiotensin II. These data showed that EFS released angiotensin II and noradrenaline from pineal slices and that a close functional connection exists between the peptide and acetylcholine. The stimulation of serotonin biosynthesis and release by these two neurotransmitters was negatively regulated by noradrenaline acting through beta-adrenergic receptors.

1-Sarcosine-8-Isoleucine Angiotensin II↗

Hydroxyindole-O-methyltransferase catalyses production of methoxyindoles in rat pineal gland dependent on the concentration of hydroxy precursors and their affinity for the enzyme.

Indole metabolites were separated by thin-layer chromatography following organ culture of rat pineal glands with tritiated tryptophan. Methoxyindole production was shown to differ substantially from results obtained when pineal glands were incubated with radiolabelled serotonin. The correlation between hydroxy- and corresponding methoxyindoles was, however, remarkably similar to previous results, and indicated that in the pineal gland, it is probable that production of methoxyindoles is dependent on the concentration of the various hydroxyindoles and their relative affinities for hydroxyindole-O-methyltransferase. It is also probable that although several forms of the enzyme may exist in the pineal gland, the catalytic sites are homogeneous in their activity.

5-Hydroxytryptophan↗