Regulation of Rat Pineal Hydroxyindole-O-Methyltransferase in Neonatal and Adult Rats

The relative importance of neural, and some nonneural, mechanisms in the control of pineal hydroxyindole-O-methyltransferase (HIOMT) activity during development and in the adult rat was studied. In neonatal rats, guanethidine-treatment, bilateral superior cervical ganglionectomy (SCGX), or exposure to constant light did not prevent the initial appearance of HIOMT activity, indicating that neural stimulation of the gland is not essential for the development of HIOMT activity. In adult rats, decentralization or removal of the SCG led to a slow fall in HIOMT activity, to about 30% of control activity, indicating that the enzyme is largely under neural control. Additionally, adrenalectomy or hypophysectomy had no effect on HIOMT activity, refuting the suggestion that adrenal and/or gonadal steroids are of major importance in the regulation of this enzyme. The fall in activity of the enzyme after SCGX or exposure to constant light probably does not represent a shift in the K_m of the enzyme nor the selective disappearance of a distinct molecular species. Similar changes in HIOMT activity and cyclic GMP responsiveness occur in response to alterations in the length of the daily dark period, adding further evidence to our earlier speculation that there may be a functional relationship between these two.     

Presynaptic and Postsynaptic Effects of Neuropeptide Y in the Rat Pineal Gland

Abstract: Neuropeptide Y is colocalized with noradrena-line in sympathetic fibers innervating the rat pineal gland. In this article we present a study of the effects and mechanisms of action of neuropeptide Y on the pineal noradrenergic transmission, the main input leading to the rhythmic secretion of melatonin. At the presynaptic level, neuropeptide Y inhibits by 45%, with an EC₅₀ of 50 n M , the potassium-evoked noradrenaline release from pineal nerve endings. This neuropeptide Y inhibition occurs via the activation of pertussis toxin-sensitive G protein-coupled neuropeptide Y-Y2 receptors and is independent from, but additive to, the α₂-adrenergic inhibition of noradrenaline release. At the postsynaptic level, neuropeptide Y decreases by a maximum of 35%, with an EC₅₀ of 5 n M , the β-adrenergic induction of cyclic AMP elevation via the activation of neuropeptide Y-Y1 receptors. This moderate neuropeptide Y-induced inhibition of cyclic AMP accumulation, however, has no effect on the melatonin secretion induced by a β-adrenergic stimulation. On the contrary, in the presence of 1 m M ascorbic acid, neuropeptide Y potentiates (up to threefold) the melatonin secretion. In conclusion, this study has demonstrated that neuropeptide Y modulates the noradrenergic transmission in the rat pineal gland at both presynaptic and postsynaptic levels, using different receptor subtypes and transduction pathways.     

Stimulus Deprivation Increases Pineal Gsα and Gβ

Denervation and other forms of stimulus deprivation cause an increase in the magnitude of subsequent responses, a phenomenon commonly referred to as denervation supersensitivity. This has been well demonstrated with the cyclic AMP response to norepinephrine in the pineal gland. In the present report, we address the question of whether stimulus deprivation alters alpha and beta subunits of the GTP binding regulatory protein that stimulates adenylyl cyclase activity (Gs). Stimulus deprivation of the pineal gland was produced by denervation (superior cervical ganglionectomy), decentralization of the superior cervical ganglia, or by exposure of the animal to continuous lighting. All increased both the alpha and beta subunits of Gs (Gs alpha and G beta) by up to fourfold, as estimated using semiquantitative western blot technology. These effects were detectable after 1 day of stimulus deprivation and were sustained for 2 weeks. The stimulatory effects of constant light-induced stimulus deprivation were also apparent by measuring cholera toxin-dependent ADP-ribosylation of Gs alpha, which revealed a four-fold increase in the amount of labeled substrate. The results of in vivo studies were confirmed with in vitro studies, which demonstrated a spontaneous increase in both Gs alpha and G beta during 72 h of organ culture. The constant light-induced increases in both Gs alpha and G beta were prevented by continuous administration of isoproterenol (0.3 mg/kg/day), supporting the suggestion that adrenergic stimulation controls the levels of Gs alpha and G beta. These studies indicate that stimulus deprivation increases both Gs alpha and G beta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Methotrexate on Biopterin Levels and Synthesis in Rat Cultured Pineal Glands

Abstract: Culture of rat pineal glands in methotrexate (0.5, 5, or 10 μM) for 6 or 24 h did not alter pineal tetrahydrobiopterin (85–90% of total biopterin in cultured glands), except for a decrease of 30% after 24 h culture in 10 μM methotrexate. However, pineal dihydrobiopterin and/or biopterin (10–15% of total biopterin) was increased by methotrexate up to 2.5-fold. Biopterin detected in the culture medium following pineal culture was also increased to a similar extent after methotrexate treatment and appeared to represent leakage of pineal dihydrobiopterin and/or biopterin. Culture of glands in 5 μM methotrexate did not alter the conversion of [U-¹⁴C]-guanosine to [¹⁴C]biopterin, suggesting that pineal tetrahydrobiopterin synthesis was not altered by methotrexate. Complete inhibition of dihydrofolate reductase activity measured in pineal homogenates was obtained following culture of glands in all concentrations of methotrexate studied. Therefore, dihydrofolate reductase and dihydrobiopterin do not appear to be involved in a major biosynthetic pathway for pineal tetrahydrobiopterin from GTP, although they may have a minor role in tetrahydrobiopterin synthesis.     

Effects of Neonatal Melatonin Administration on the Extra-Hypothalamic Regions in Rat Brains: Effects on the Serotonergic System

The effects of 100g melatonin injection at postnatal day 5 (PD 5) on the development of the central serotonergic systems in male and female rats were investigated. The contents of serotonin (5-HT) and 5-hydroxy-3-indolacetic acid (5-HIAA) were measured in several extra-hypothalamic regions at 3, 10 and 42 weeks of age. The neonatal melatonin administration increased both 5-HT and 5-HIAA levels in the striatum throughout the examined period. In the hippocampus, an increase in 5-HIAA contents by neonatal melatonin administration was found at 3 weeks but not 10 or 42 weeks of age. There were no significant differences in the effects of melatonin between male and female rats. These results indicated that exogenous melatonin administration during the early neonatal period influenced the development of the serotonergic systems in extrahypothalamic regions including the hippocampus and the striatum.     

Early Effects of Neonatal Rat Desympathization on Secondary Heart Rhythms

Neonatal Wistar rats for the first 3 weeks of life were injected intraperitoneally with isobarine every other day. The single doze was 40 mg/kg. Control animals were injected with saline. Degenerative changes in sympathetic ganglia were evident as early as in the 10-day old animals and increased by 18–19 days. The heart rate in the desympathized animals was lower than in control from 10–11 to 18–19 days, but by the end of the 3rd week the differences were eliminated. The same occurred with respiration rate. At same terms there was an essential decrease of amplitude of the heart rate high-frequency fluctuations synchronous with respiration and of the periodogram slow waves with the period about 1 min. Using the method of fast Fourier transform, the power spectra of heart rate fluctuations (secondary heart rhythms) in 5 frequency ranges (0–0.01, 0.01–0.03, 0.03–0.1, 0.1–1.0, and 1.0–2.5 Hz) were calculated. Desympathization leads to a decrease of the fluctuation power in all ranges, but in the ultralow-frequency range this decrease is the least pronounced, which suggests the presence of non- sympathetic mechanisms in their genesis. The greatest changes occur in the middle-frequency area. In all cases, differences from control values increase from the 10–11th to the 18–19th days, after which a tendency for restoration is observed, in spite of an enhancement of processes of degeneration of sympathetic neurons. This indicates an activation of the compensatory mechanisms, due to which consequences of desympathization are partially smoothed at distant terms of studies.     

赤芍总苷对培养乳鼠心肌细胞损伤的保护作用

为了探讨赤芍总苷对心肌细胞损伤的保护作用,以异丙基肾上腺素加入培养的乳鼠心肌细胞造成缺血缺氧损伤模型,对比分析正常对照组、药物损伤组、辅酶Q10阳性对照组、以及不同剂量赤芍总苷保护组的细胞形态学、心肌酶谱等指标、结果显示：损伤组细胞搏动加速,存活率下降,GOT、LDH、CK等3种心肌酶活力均显著升高;而辅酶Q10组和赤芍总苷组上述指标都有不同程度的改善,其中高剂量赤芍总苷组的保护作用优于阳性对照组．证明赤芍总苷对异丙基肾上腺素造成的培养乳鼠心肌细胞损伤具有保护作用,并且呈现剂量依赖关系．     

Lipoxygenation of Docosahexaenoic Acid by the Rat Pineal Body

Abstract: Based on the inhibitor profile, production rate, and stereochemical purity of the hydroxylated products, it was demonstrated that lipoxygenation in rat brain occurs only in the pineal. Both positional and stereochemical specificities of the hydroxylation were observed only in pineal, clearly indicating that only the pineal is capable of lipoxygenating polyunsaturated fatty acids among the rat brain regions examined. Cerebral cortex also produced hydroxy products; however, they were racemic mixtures, indicating that peroxidation was responsible for their production. Rat pineal homogenate, obtained after the brain was perfused, metabolized [¹⁴C]docosahexaenoic acid ([1–¹⁴C]22:6n3) to monohydroxy derivatives, primarily by the 12-and, to a lesser extent, by the 15-lipoxygenase (LO) reaction. The resulting metabolites were 14(S)-and 17(S)-hydroxydocosahexaenoic acid (HDoHE), as determined by reversed-phase HPLC, chiral-phase HPLC, thermospray liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry. Because blood was removed by perfusion of the brain before incubation, it was clear that the observed LO activity was not due to contamination with blood cell components. The production rate of 17-HDoHE from 22:6n3 was higher than that of 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid from 20:4n6, whereas 12-LO activity toward these two substrates was comparable. These monohydroxy metabolites were also detected in the pineal body lipid extract using negative ion chemical ionization mass spectrometry. This is the first observation of endogenous production of hydroxylated compounds in pineal. The ratio of endogenous 15-LO to 12-LO products was considerably higher than that of the in vitro production from exogenous substrate. In some cases, 15-LO products were the major LO metabolites present in the lipid extract of pineal body for both 20:4n6 and 22:6n3. Both 12-and 15-LO activities were recovered mainly in the microsomal plus cytosolic fraction. In addition to monohydroxy products, epoxy, hydroxy derivatives were formed from 22:6n3 by the pineal. The major isomer was identified as 12-hydroxy-13, 14-epoxy-22:5n3. Key Words : Lipoxygenation-Docosahexaenoic acid (22:6n3)—Pineal—Rat brain—Hydroxydocosahexaenoic acid—Hepoxilin-like compound.     

Adrenergic Control of Rat Pineal NO Synthase

Abstract: We have previously shown that exposure of rats to constant light (LL) induced a decrease in NO synthase (NOS) activity in the pineal gland. We present here the evidence that chronic (5 days) norepinephrine (NE) or isoproterenol treatment prevents the effect of LL and enhances pineal NOS activity in LL animals. This effect of NE appears to be mediated by β-adrenoceptors, because it was not mimicked by the α-agonist phenylephrine. Pineal NOS activity was reduced in 16-h light/8-h dark animals treated for 4 days with the β-adrenergic antagonist propranolol but not with the α₁-antagonist prazosin, indicating again an involvement of β-adrenergic receptor in the control of NOS. Treatment with adrenergic antagonists did not affect cortical NOS activity, suggesting that the control of NOS is different in these two tissues or that the pineal expresses a specific isoform of the enzyme. Taken together, these data suggest that NE controls NOS in the pineal gland through β-adrenergic receptors. To our knowledge, this represent the first demonstration of a regulation of NOS by a neurotransmitter in the CNS, as assayed under V _max conditions.     

Selective Effects of Neonatal Hypothyroidism on Monoamine Oxidase Activities in the Rat Brain

Hypothyroidism of mild intensity was obtained with prenatal and neonatal submission of Long-Evans rats to an iodide-rich diet. Chronic daily administration of methimazole to iodide-supplemented Long-Evans pups or to iodine-deprived Charles-River rats through the first 29–30 days of age provoked severe hypothyroidism. Monoamine oxidase type A (MAO-A) and not type B (MAO-B) activity was consistently, although slightly (by approximately 20%), increased in the hypothyroid brain. Triiodothyronine (T₃)-induced hyperthyroidism did not affect MAO activity. Replacement therapy with T₃ did not normalize MAO-A activity in hypothyroidism. Methimazole displayed a competitive and reversible in vitro inhibition of MAO-A but not MAO-B activity. Although this effect was obtained at concentrations far higher than those estimated to reach the brain after a single injection of the goiterogen, the occurrence of accumulation processes in the metabolism-deficient hypothyroid neonate rs cannot be excluded. Thus, MAO-A activity might be either directly depressed during the goiterogenic treatment, or increased as the result of some kind of rebound effect after interruption of methimazole administration.     

Neonatal Hormone Treatment and Maturation of the Pineal Noradrenergic System: Hydrocortisone and Thyroxine

Abstract The circadian release of norepinephrine from nerve terminals in the pineal gland drives acetyl-CoA:serotonin N -acetyltransferase (NAT; EC 2.3.1.5) activity in the adult pineal from a daytime low to a nighttime high. In the newborn, enzyme activity is intermediate between the adult's daily extremes and has only a small circadian fluctuation. With age, these fluctuations increase in amplitude until the adult pattern is attained at about days 10–12. Treatment of neonates with thyroxine for the first 3 days of life accelerated, whereas administration of hydrocortisone acetate at birth retarded the developmental decline in daytime serotonin-N-acetyltransferase activity. Maximal differences in daytime enzyme activity of controls and thyroxine-treated animals were seen at day 4 and between controls and steroid-treated pups at day 8. Desipramine treatment increased NAT activity in 8-day-old animals; hydrocortisone-treated animals were least affected. Freshly cultured pineals from steroid-treated animals were more responsive to low, and less responsive to high, concentrations of norepinephrine than glands from thyroxine-treated or control animals. They were also less responsive to isoproterenol both in acute and 48-h organ culture. Pineals from hydrocortisone-treated animals in culture accumulated less exogeneous norepinephrine than glands from controls but released a greater fraction of their content on transfer to fresh medium. Normal and steroid-treated animals released the same fraction of their norepinephrine contents into the medium when reuptake was blocked by desipramine (DMI).     

Regulation of Rat Pineal Hydroxyindole-O-Methyltransferase: Evidence of S-Adenosylmethionine-Mediated Glucocorticoid Control

: Rat pineal hydroxyindole-O-methyltransferase is controlled similarly to adrenal medullary phenylethanolamine N-methyltransferase. S-adenosylmethionine (SAM), the in vivo cofactor utilized by the enzyme to convert N-acetylserotonin to melatonin, protects this methyltransferase against tryptic proteolysis in vitro. Furthermore, in vivo studies suggest that the nucleoside itself is controlled by glucocorticoids. Hypophysectomy decreases hydroxyindole-O-methyltransferase levels as compared with control animals, while dexamethasone and SAM administration restore enzyme levels toward control values. In vitro proteolytic studies further demonstrate that, although N-acetylserotonin does not stabilize the enzyme against trypsinization, this substrate acts synergistically with SAM to confer greater stabilization than observed with SAM alone.     

Circadian Variation of Cyclic AMP in the Rat Pineal Gland

Abstract: This study was carried out to investigate circadian variation of cyclic AMP contents in the rat pineal glands, using the high-energy microwave radiation technique. The pattern of cyclic AMP concentration in the pineal gland showed a distinct circadian variation, with the maximum level at 0200 and the lowest at 1400. The administration of propranolol completely blocked the dark-induced increase in the pineal cyclic AMP level at 0200, and the administration of isoproterenol induced a threefold, rapid increase in the cyclic AMP level at 1400, although it did not change the level at 0200.     

Reproductive Effects of the Mammalian Pineal

The reproductive systems of mammals are anatomically and physiologicallyaltered by the pineal gland. Characteristically, pineal removalaccelerates gonadal growth in immature animals and causes atransient modest enlargement of the reproductive organs if theoperations are done after adulthood is attained. Light deprivationrenders the pineal strongly antigonadotropic. In addition, thereare several conditions, e.g., androgen-sterilization and anosmia,which potentiate, probably indirectly, the gonad-inhibitingcapability of the rat's pineal. The chronic administration ofpineal extracts or pineal substances has effects opposite topinealectomy, i.e., they restrict gonadal development or growth.The pineal is possibly important in adjusting the level of reproductiveactivity to seasonal changes in environmental conditions.     

Photoneural Regulation of Rat Pineal Nitric Oxide Synthase

Abstract: We report here a photoneural regulation of nitric oxide synthase (NOS) activity in the rat pineal gland. In the absence of the adrenergic stimulation following constant light exposure (LL) or denervation, pineal NOS activity is markedly reduced. A maximal drop is measured after 8 days in LL. When rats are housed back in normal light-dark (LD) conditions (12:12), pineal NOS activity returns to normal after 4 days. A partial decrease in pineal NOS activity is also observed when rats are placed for 8 days in LD 18:6 or shorter dark phases, indicating that pineal NOS activity reflects the length of the dark phase. Because it is known that norepinephrine (NE) is released at night from the nerve endings in the pineal gland and this release is blocked by exposure to light, our data suggest that NOS is controlled by adrenergic mechanisms. Our observation may also explain the lack of cyclic GMP response to NE observed in animals housed in constant light.     

Microdialysis of Melatonin in the Rat Pineal Gland: Methodology and Pharmacological Applications

Abstract: 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 fluori-metric 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 β-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 β-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 α₁-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.