[Recent aspects on the physiology and biochemistry of the pineal gland. II. Biological properties of the pineal gland].
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Adrenal Cortex Hormones↗
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The pineal gland of nocturnal mammals. II. The ultrastructure of the pineal gland in the pipistrelle bat (Pipistrellus pipistrellus L.): presence of two populations of pinealocytes.
In the pineal gland of the pipistrelle bat two different populations of pinealocytes and glial cells were observed electron microscopically. The pinealocytes of populations I and II differ in their content of metabolically active cell organelles. In the pinealocytes of population I, granular vesicles originating from the Golgi apparatus were found in the perikaryon and especially in the endings of the pinealocyte processes. Granular vesicles appeared to be more numerous in hibernating nulliparous females. The pinealocytes of population II are characterized by the presence of small cytoplasmic vacuoles, probably originating from cisternae of the granular endoplasmic reticulum and containing flocculent material of moderate electron density. The classification of the pinealocytes belonging to population II is discussed.
Animals↗
Catecholamine receptors regulating serotonin N-acetyltransferase activity and melatonin content of chicken retina and pineal gland: D2-dopamine receptors in retina and alpha-2 adrenergic receptors in pineal gland.
The type of catecholamine receptor involved in the regulation of serotonin N-acetyltransferase (NAT) activity and melatonin (N-acetyl-5-methoxytryptamine) level in chicken retina was compared to that regulating these parameters in the pineal gland. Systemic administration of apomorphine, a dopamine receptor agonist, resulted in marked inhibition of the nocturnal increase of retinal NAT activity and melatonin content. Apomorphine did not affect NAT activity or melatonin content of the pineal gland. In contrast, clonidine, an alpha-2 adrenergic receptor agonist, inhibited the nocturnal rise in pineal NAT activity and melatonin content although being without effect on these parameters in retina. Apomorphine-induced inhibition of retinal NAT activity was blocked by spiperone, a D2-dopamine receptor antagonist, but not by antagonists of D1-dopamine, alpha-1, alpha-2 and beta adrenergic receptors. Systemic or intraocular injection of quinpirole, a D2-dopamine receptor agonist, in the middle of the dark phase of the light-dark cycle markedly reduced retinal NAT activity and melatonin level, whereas injections of SKF 38393-A, a D1-dopaminergic agonist, had no effect. The inhibitory effect of clonidine on pineal NAT activity was blocked by yohimbine, an alpha-2 adrenergic receptor antagonist. The results presented in this paper demonstrate that NAT activity and melatonin content in chicken retina and pineal gland are differentially modulated in vivo by D2-dopamine and alpha-2 adrenergic receptors, respectively. Despite the different types of receptors involved, both tissues may share a common pathway for catecholamine-mediated inhibition of melatonin biosynthesis, i.e., inhibition of adenylate cyclase activity.
Acetyltransferases↗
An antiserum against chicken hydroxyindole-O-methyltransferase reacts with the enzyme from pineal gland and retina and labels pineal modified photoreceptors.
Biosynthesis of the indolic hormone melatonin has been reported in the pineal gland and retina. The terminal step of melatonin synthesis is catalysed by hydroxyindole-O-methyltransferase (HIOMT), an enzyme displaying highest levels of activity in the pineal gland and retina. Several laboratories have suggested that melatonin synthesis might take place in retinal photoreceptors and in photoreceptor-derived cells of the pineal gland. Experimental support to this hypothesis is progressively building up with the immunocytochemical identification of HIOMT-containing cells in various animal species. In the present report, HIOMT was purified from the chicken pineal gland using a one-step chromatographic procedure and an antiserum against the enzyme was obtained in the rabbit. The antiserum was further purified by immunoadsorption chromatography on chicken brain proteins. Using electroblots immunochemical labeling, HIOMT from chicken pineal gland and retina was identified as a 38-kDa protein. Pineal HIOMT was further resolved into components of different pHi-values (5.4-5.7 and 6.8), using two-dimensional gel electrophoresis. Immunoprecipitation of HIOMT activity was observed in pineal homogenates and, for the first time, in homogenates of the retina. Immunofluorescence microscopy provided the first evidence that HIOMT is contained in modified photoreceptors of the chicken pineal gland. No immunofluorescence could be observed in the retina, maybe due to the lower level of HIOMT activity in this tissue. Together, the data indicate that the antiserum may be a useful tool to study the regulation of HIOMT synthesis in the pineal gland and in the retina. Further work is required to identify HIOMT-containing cells in the retina.
Acetylserotonin O-Methyltransferase↗
Status of dopamine in bovine pineal glands and the stimulation of N-acetyltransferase activity by D2-dopaminergic receptor agonists in the rat pineal glands in culture.
In a previous study, we identified in the bovine pineal gland two [3H]spiroperidol-binding sites with KD values of 0.18 and 2.1 nM and Bmax values of 37 and 630 fmol/mg protein, respectively. In this study, the status of dopamine in the bovine pineal glands was delineated further by measuring the relative concentrations of dopamine and norepinephrine and the relative concentrations of serotonin and melatonin. Furthermore, the presence of 4.0 +/- 0.6 micrograms/dopamine/gm tissue encouraged us to delineate the effects of select dopaminergic receptor agonists and antagonists on the synthesis of melatonin in vivo and on the activity of N-acetyltransferase in the rat pineal gland in culture. The acute administration of haloperidol (3 mg/kg intraperitoneally [ip]) or sulpiride (200 mg/kg ip) increased the concentration of melatonin in the pineal gland from 160.6 +/- 8.18 to 327.6 +/- 45.43 and 306.5 +/- 40.53 pg/gland, respectively. Dopamine exhibited dual effects on the activity of N-acetyltransferase, inhibiting the basal activity at 0.1 microM and stimulating it at 10 microM, and the later effect was blocked by propranolol. D2-dopaminergic receptor agonists such as bromocriptine (4.0 microM) or LY-171555 (10.0 microM) partially attenuated the norepinephrine-induced stimulation of N-acetyltransferase, and these attenuating effects were reversed by D2-dopaminergic antagonists such as haloperidol (10 microM) or domperidone (10 microM). The results of these studies are interpreted to indicate that for the synthesis of melatonin, the pineal D2-dopaminergic receptors may function independently from those of the beta 1-adrenergic receptor sites. Furthermore, the said D2-dopaminergic receptor are amenable to down regulation since the activity of N-acetyltransferase remained unaltered (0.0717 vs. 0.0729 nmol/gland/h) following chronic treatment (4 mg/kg ip/day for 30 days) with bromocriptine.
Acetyltransferases↗
Structure and ultrastructure of the aging rat pineal gland.
The pineal gland of rats 12-28 months old was studied with light and electron microscopes. All pineal components exhibited regressive changes of different intensity with age. In type I pinealocytes, there was a marked increase in dense bodies as well as the occasional appearance of wide cell profiles full of vesicles. Type II pinealocytes showed nuclear infoldings and cytoplasmic deposits of lipofuscin. Pineal stroma displayed an increase in connective tissue fibers, both collagen and oxytalan, as well as remains of basement membranes and other materials of unknown nature. Calcareous concretions were also found, mostly in the pineal capsule. All regressive changes were more intense with increasing age.
Aging↗
The pineal gland of the gerbil, Meriones unguiculatus. III. Morphometric analysis and fluorescence histochemistry in the intact and sympathetically denervated pineal gland.
Morphometric analytical procedures were employed to study the pineal gland of the Mongolian gerbil following superior cervical ganglionectomy (SCGX). The purpose of this study was to define the effects of sympathetic denervation on the morphology of the gland at two time periods, 0500 and 1900 h (one hour before lights-on and lights-off, respectively). Fluorescence histochemistry was employed to determine catecholamine and indoleamine content in intact and denervated pineal glands. After SCGX, the pinealocytes decrease in size, concretions are prevented from forming, and the yellow fluorescence in the gland is lost. Following denervation a depression in the volume of most of the pinealocyte organelles, i.e., SER, RER/ribosomes, free cytoplasm, mitochondria and presumptive secretory vesicles, was also observed. However, synaptic ribbons increased in volume in the gerbils that had been killed at 1900 h. It appears that the sympathetic innervation to the pineal gland is a requirement for the presumptive secretory activity of the pinealocytes.
Animals↗
The effect of castration, testosterone and estradiol on 14C-serotonin metabolism by organ cultures of male rat pineal glands.
The pineal gland of the male rat does not appear to rely on prior conversion of testosterone to estradiol for the stimulant effect of testosterone on pineal melatonin and 5-methoxytryptophol synthesis.
Animals↗
Vasopressin uptake by hypothalamopituitary axis and pineal gland in guinea pigs.
The uptake of circulating arginine vasopressin (AVP) by the pituitary gland (anterior lobe), pineal gland, and hypothalamus (ventromedial part) was investigated in an isolated in situ perused brain of anesthetized guinea pig. Kinetic experiments revealed saturable AVP uptake in all studied regions with Km values of 0.79, 0.19, and 0.76 microM and maximum velocity values of 22, 2.1, and 1.6 pmol.min-1.g-1 for the pituitary gland, pineal gland, and hypothalamus, respectively. The nonsaturable components (diffusion constants) were not significantly different from zero. Peptide fragments, L-phenylalanine, and Bestatin (an aminopeptidase inhibitor) did not interfere with AVP uptake. However, uptake of AVP was strongly inhibited in the presence of the V1 antagonist [1-(beta-mercapto-beta-beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine]AVP at 2.7 microM, and regional Ki values, assuming that the observed inhibitions were purely competitive, ranged between 0.32 and 2.23 microM. The V2 agonist 1-desamino-8-D-AVP at 2.7 microM produced only a weak inhibition of AVP uptake, and regional Ki values ranged between 9.56 and 21.3 microM. It is concluded that specific uptake mechanisms in the hypothalamopituitary axis and pineal gland are sensitive enough to detect blood-borne AVP under the physiological hormonal state. It is suggested that AVP binding in situ is primarily related to V1 receptors, which may be involved in mediating the central effects of this circulating peptide.
Animals↗
Vasopressin potentiation of the melatonin synthetic pathway via specific V1a receptors in the rat pineal gland.
The pineal gland releases the "time-keeping' hormone melatonin following a rhythmic sympathetic input which translates light information. The aim of this work was to study the role and mechanism of action of the central vasopressinergic input on pineal cAMP-dependent melatonin synthesis in the rat. The pineal was found to display vasopressin receptors of the V1a subtype, as the V1a antagonist [125I]HO-LVA bound in a saturable manner to pineal membranes with a high affinity (kd = 10 pM) and a maximal binding capacity (B(max)) of 13 fmol/mg protein. Vasopressin was able to displace [125I]HO-LVA binding in a dose-dependent manner (k(i) = 1.9 nM). Vasopressin had no effect on the basal cAMP level and melatonin secretion in cultured rat pinealocytes. However, it clearly and dose-dependently (EC50 = 7 nM) potentiated by 2-3 times cAMP accumulation and by 1.5-2.5 times melatonin secretion induced by moderate noradrenergic stimulation. On strongly stimulated pinealocytes, however, vasopressin could potentiate cAMP accumulation, but not melatonin secretion. The potentiatory effect of vasopressin was inhibited in the presence of the V1a antagonist. These results indicate that vasopressin is a potent modulator of rat pineal synthetic activity.
Amino Acid Sequence↗
Precise annual changes in the numbers of "synaptic" ribbons and spherules in the rat pineal gland.
Although pineal "synaptic" ribbons (SR) are frequently examined by means of quantitative electron microscopy, their functional significance remains unclear. The same is true for related structures--"synaptic" spherules (SPH). In the course of such studies, it has been noted that SR counts may differ from laboratory to laboratory. Because seasonal changes may play a role, a 2-year study was performed on male rats kept under routine laboratory conditions and killed at monthly intervals during daytime or nighttime. Both structures examined showed distinct day-night differences throughout the year, with higher numbers at night than during the day. There were significant annual changes in both SR and SPH during both daytime and nighttime. The comparison of the curves from the 2 years showed that they were virtually identical both during daytime and nighttime. The numbers of SR were the highest in October and the lowest in April; the numbers of SPH had two plateaus, one with lower values from November to April, and the other with higher values from May to October. It appears from the present study that SR and SPH numbers in the rat pineal gland show statistically significant and precisely timed seasonal changes that may well account for the variations of SR numbers in the different publications.
Animals↗
Adrenergic and peptidergic control of the regulation of cAMP efflux and melatonin secretion from perifused rat pineal gland.
Mammalian pineal gland receives peptidergic (e.g., vasoactive intestinal peptide [VIP]; peptide histidine isoleucine [PHI]; neuropeptide Y, NPY; substance P, calcitonin gene-related peptide [CGRP], arginine vasopressin [AVP] and oxytocin [OXT]) fibers in addition to sympathetic innervation. The dynamics of cAMP efflux and melatonin (MT) secretion were compared during the infusion of these peptides in our long-term perifusion system. VIP and PHI enhanced both pineal cAMP efflux and MT secretion in a dose-dependent manner (10 nM to 10 microM). However, the potency of PHI was slightly less. The peak of cAMP release always precedes that of MT production. The possible interactions between adrenergic and peptidergic compounds in the regulation of pineal cAMP efflux and MT secretion were also studied. VIP acts on specific peptidergic receptors, since its stimulatory effect could only be reduced by a VIP receptor antagonist. VIP has an additive effect at a lower (100 nM) concentration combined with norepinephrine (NE). NPY (100 nM) can completely block NE-induced MT secretion, but the decrease in cAMP efflux is less. However, NPY does not significantly influence VIP-stimulated cAMP efflux or MT secretion. These data suggest that NE, VIP, and NPY are differently involved in the cAMP and calcium signaling. The other neuropeptides are ineffective.
Animals↗
In vitro effects of putative neurotransmitters on synaptic ribbon numbers and N-acetyltransferase activity in the rat pineal gland.
The pineal contains a large number of classical transmitters and neuropeptides. Some of these neurochemicals are involved in the regulation of serotonin N-acetyltransferase (NAT) activity and hence in melatonin synthesis. Synaptic ribbons present in the pineal gland also exhibit a numerical day/night rhythm parallel to that of NAT activity. There is scarcity of information regarding the regulation of synaptic ribbon (SR) numbers. In the present study, we have investigated in vitro effects of a number of classical neurotransmitters and neuropeptides. NAT activity was used to monitor melatonin synthesis under the experimental conditions used. Norepinephrine (NE), Delta sleep-inducing peptide (DSIP), vasoactive intestinal polypeptide (VIP), adenosine and N-acetyl-asp-glu (NAAG) significantly increased NAT activity in rat pineal. DSIP and VIP also increase the stimulatory effect of NE on NAT activity. These neurochemicals had no effect on SR numbers. Gamma aminobutyric acid (GABA), serotonin and taurine affected neither NAT activity nor SR. Somatostatin increased SR numbers significantly, without having any effect on NAT activity. The effect of somatostatin is regarded to be pharmacologic, since rather high dosages (10(-4) M) were required to obtain a significant effect. Although somatostatin is present in the pineal and may change rhythmically, the inconsistency of the day/night rhythmicity and the lack of such a rhythm in female rats and male gerbils speaks against an important physiological role of somatostatin in regulating SR numbers.
Animals↗
Fine structural alterations in the aging rat pineal gland.
The pineal glands of 1 mo to 28 mo old male and female rats were examined by electron microscopy. With increasing age the following observations were made: (1) the capsule thickness increases; (2) there is an increase in collagen infiltration; (3) increased amounts of granular deposits are seen between cells; (4) there is a greater variability in the number of light pinealocytes; (5) pinealocytes with nuclear invaginations and pinealocytes with nuclear inclusions increase in number; (6) more cytoplasmic dense bodies are seen in pinealocytes and gliocytes; (7) the maximum diameter of pinealocyte lipid droplets tends to increase; (8) no definite change is observed in granular endoplasmic reticulum; (9) occasional cells are found to contain reticulated mitochondria, and a few cell processes have an appearance similar to neuroaxonal dystrophy. Besides illustrating age-rated changes, the present study proves the existence of pinealocyte nuclear inclusions, suggests the possibility of pineal concretions in the rat, and morphometrically or semi-morphometrically analyzes several structural features not previously quantified.
Aging↗
Effect of oxygen on the induction of tryptophan hydroxylase by adrenergic agents in organ cultures of rat pineal glands.
Rat pineal glands, cultured under 95% air and 5% CO2, lost 50% of their tryptophan hydroxylase activity within 5 h. This loss was accelerated by the addition of cycloheximide or puromycin to the medium. Activity was, however, largely maintained in 95% O2 and 5% CO2. Under these conditions, L-noradrenaline (100 microM), L-isoproterenol (10 microM), and dibutyryl cyclic AMP (1 mM) induced enzyme activity. They failed to do so when 95% air and 5% CO2 was used. Noradrenaline induced serotonin N-acetyltransferase activity with either atmosphere.
Animals↗
Circadian and photic regulation of MAP kinase by Ras- and protein phosphatase-dependent pathways in the chick pineal gland.
Chick pineal mitogen-activated protein kinase (MAPK) exhibits circadian activation and light-dependent deactivation at nighttime. Here we report that, in the chick pineal gland, levels of active forms of MAPK, MEK, Raf-1 and Ras exhibited synchronous circadian rhythms with peaks during the subjective night, suggesting a sequential activation of components in the classical Ras-MAPK pathway in a circadian manner. In contrast, the light-dependent deactivation of MAPK was not accompanied by any change of MEK activity, but it was attributed to the light-dependent activation of protein phosphatase dephosphorylating MAPK. These results indicate that the photic and clock signals regulate MAPK activity via independent pathways, and suggest a pivotal role of MAPK in photic entrainment and maintenance of the circadian oscillation.
Animals↗
Immunoreactive luteinizing hormone-releasing hormone (LHRH) in ovine pineal glands.
Ovine pineal glands (PGs, n = 168), cerebral cortex(C) and stalk-median eminences (SMEs) were obtained fresh from slaughterhouse sheep and mature ewes during the estrous cycle. Tissues were extracted in 2N acetic acid (aa) followed by either glacial aa or ethanol. Portions of the preparations were further subjected to gel filtration using Sephadex G-25. Neutralized extracts were assayed for LHRH using radioimmunoassay. LHRH immunoreactivity was consistantly detected in the various extracts, ranging from 7-150 pg/PG (0.1-1.7 pg/mg), 0.1-0.3 pg/mg C and 35-72 ng/SME (0.7-1.4 ng/mg). Slopes of binding displacement regression lines for the preparations were not different from that of synthetic LHRH. Chromatographic elution profiles for preparations of SMEs and PGs were similar and the bulk of immunoreactivity eluted in a position coincident with that of synthetic LHRH. Immunoreactivity in cortical extracts ran in advance of the others. PG contents of LHRH immunoreactivity were similar during the estrous cycle.
Animals↗
Photoneural regulation of the mammalian pineal gland.
Mammalian pineal function appears to be controlled primarily through the release of noradrenaline from the terminals of nerves whose cell bodies lie in the superior cervical ganglia. This is the final segment of the following neural pathway: retina----retinohypothalamic projection----suprachiasmatic nuclei----paraventricular nuclei----intermediolateral cell column----superior cervical ganglia----nervi conarii----pineal gland. Noradrenaline acts on pinealocytes through alpha- and beta-adrenoceptors in an atypical manner. Beta-Adrenergic activation is an absolute requirement for the stimulation of both cyclic AMP and cyclic GMP production, and by itself produces a sixfold increase in the former and a twofold increase in the latter. Alpha-Adrenergic activation potentiates the beta-adrenergic stimulation of cyclic AMP production 10-fold, and that of cyclic GMP production about 200-fold. The mechanism of alpha- and beta-adrenergic interaction is being examined, and progress is being made in understanding the adrenergic control of cyclic AMP. It appears that alpha-adrenergic agonists act through the alpha 1-subclass of adrenoceptors to stimulate phospholipid turnover and the production of a breakdown product of phosphatidylinositol, diacylglycerol. This compound promotes the association of protein kinase C with membranes, which leads to the marked phosphorylation of one protein. The precise identity of this protein remains a mystery. This interaction leads to a larger cyclic AMP response but does not appear to be involved in the mechanism of potentiation of the cyclic GMP response. Changes in chronic neural stimulation produce reciprocal changes in the magnitudes of cyclic AMP and cyclic GMP responses. Chronic denervation results in a supersensitive cyclic AMP response and nearly complete disappearance of the cyclic GMP response. This is termed 'see-saw' signal processing. All the available evidence indicates that melatonin production is regulated by cyclic AMP. This nucleotide not only increases the activity of serotonin N-acetyltransferase (more correctly called arylalkylamine N-acetyltransferase) but also stabilizes the enzyme and prevents its inactivation.
Animals↗