Determination of six indolic compounds, including melatonin, in rat pineal using high-performance liquid chromatography with serial fluorimetric-electrochemical detection.

A reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, N-acetylserotonin, tryptophan, 5-hydroxytryptamine (serotonin) and melatonin in rat pineal using a buffered aqueous eluent containing acetonitrile and methanol as organic modifiers and an ion-pairing agent to assist in controlling the retention of compounds containing an amine group. Serial fluorimetric-electrochemical detection provided additional assurance of compound identity. Analyte preparation simply involved the sonication of pineals in dilute perchloric acid containing an antioxidant and a chelating agent, followed by centrifugation to clarify. The method simplifies the determination of this range of indolic compounds, which normally would require at least two separate runs with different eluents. Detection limits for melatonin were 60 and 135 pg for fluorimetric and electrochemical detection, respectively. (This represented the "worst case" considering the levels and detection limits of all compounds present.) Using flow programming and a flow-rate varying between 1 and 1.5 ml/min, the analysis time was 27.5 min, which made the determination of ten samples in a working day possible.

Characterization of D1 dopamine receptors in the bovine pineal gland with [3H]SCH 23390.

Previous studies from this laboratory have shown that bovine pineal glands not only contain D2 dopamine receptors, but also that dopamine has dual effects on the activity of pineal serotonin N-acetyltransferase, inhibiting the basal activity at 0.1 microM and stimulating it at 10 microM. We report here that the bovine pineal gland also possesses D1 dopamine receptors, which were characterized by using [3H]SCH 23390, the selective D1 dopamine receptor antagonist. SCH 23390 bound to bovine pineal membranes in a stereoselective, saturable and reversible manner. The receptor density (Bmax) was 974 fmol/mg protein and the dissociation constant (KD), as determined by saturation studies, was 0.56 nM, which is in good agreement with the KD calculated from kinetic studies (0.40 nM). Dopaminergic receptor agonists and antagonists inhibited [3H]SCH 23390 binding in a concentration-dependent and stereoselective fashion, demonstrating a pharmacology characteristic of D1 dopamine receptors. On the other hand, serotonin and serotonin receptor antagonists had a very low potency in inhibiting [3H]SCH 23390 binding. Studies involving dopamine receptor agonists revealed a low density, high affinity binding site (10%) and a high density, low affinity binding site (90%) in the bovine pineal gland. The results of this study are interpreted to indicate that the effects of dopamine in the pineal gland are modulated through mechanisms that involve both D1 and D2 dopamine receptors.

Immunohistochemical localization of synaptophysin (p38) in the pineal gland of the Mongolian gerbil (Meriones unguiculatus).

Synaptophysin (protein p38), a major integral membrane glycoprotein of small presynaptic vesicles, was localized immunohistochemically in semithin sections of the superficial pineal gland of the Mongolian gerbil (Meriones unguiculatus). Synaptophysin immunoreactivity could be detected in all pinealocytes, which were visualized with antibodies directed against neuron-specific enolase (NSE) in adjacent sections. No p38 immunoreactivity was discernible in the interstitial glial cells, which showed a heterogeneous pattern of immunostaining for the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Pinealocytes exhibited considerable intercellular differences in the densities of immunostaining. The various degrees of synaptophysin immunoreactivities in pinealocytes were not correlated with the densities of NSE immunostaining. Nerve terminals and varicosities displayed stronger immunoreactivities than pinealocytes. They were particularly numerous in the perivascular spaces. It is not clear whether this distribution indicates an innervation of pineal capillaries in addition to the functionally important innervation of pinealocytes. Several highly p38-positive dots of variable size were a conspicuous feature throughout the gland. By the consecutive semithin-thin section technique, they could be identified as processes of pinealocytes, filled with accumulations of small clear vesicles. Obviously, these vesicles represent the major site of synaptophysin immunoreactivity in pinealocytes. In the gerbil, similar vesicles have been ascribed a role in the secretory activity of the gland, and/or in the transport of calcium. The intercellular differences in the degrees of p38 immunostaining may, therefore, reflect different states of a specific cellular activity. The presence of synaptophysin in pinealocytes of the normal pineal, including the deep portions of the gland, emphasizes the paraneuronal character of these cells.

Determination of pineal melatonin by high-performance liquid chromatography with electrochemical detection: application for rhythm studies and tissue explants.

High-pressure liquid chromatography with electrochemical detection has been used for the determination of melatonin in rat pineal glands and tissue explants. Rat pineal melatonin content was measured at 2-hr intervals during a 24-hr period by direct injection of supernatants from centrifuged pineal homogenates into a reversed-phase C18 column. The lower limit of sensitivity was 32 pg per gland for a 2:1 signal-to-noise ratio, which allows melatonin measurements during daytime. Both absolute values and circadian rhythmicity obtained with the present method are in agreement with previously validated radioimmunoassays. In addition, isoproterenol-induced release of melatonin from individual rat pineal explants was measured after chloroform extraction of the incubation medium.

Vasoactive intestinal peptide-like immunoreactive nerve fibers in the pineal gland of the sheep.

Vasoactive intestinal peptide (VIP)-like immunoreactive nerve fibers were demonstrated by peroxidase antiperoxidase (PAP) immunohistochemistry to be distributed throughout the entire pineal gland of the sheep. VIP-containing fibers were observed along the blood vessels, penetrating into the gland from the pial capsule and also in the capsule itself. Some fibers left the perivascular position and entered the pineal parenchyma, where they were located among pinealocytes. This suggested that the VIPergic fibers might influence both pinealocytes and blood vessels of the gland. The location of VIP-containing fibers in the capsule of the pineal gland indicates that the fibers originate from perikarya located in a peripheral ganglion.

Neuronal markers in the rodent pineal gland--an immunohistochemical investigation.

Although some embryological and morphological features speak in favour of a neuronal character of rodent pinealocytes, histochemistry and ultrastructure let this issue appear controversial. Using antibodies to different neurofilaments, the neural adhesion molecule L1, synaptophysin and tubulin as neuronal markers, the pineal glands of rat and guinea-pig were studied by means of immunofluorescence. Neurofilament-immunoreactivity was present in some rat pineal nerve fibers and in the majority of guinea-pig pinealocytes, L1 decorated rat intrapineal nerve fibers, synaptophysin was almost ubiquitously distributed in the pineal of both species, while tubulin-immunofluorescence was seen in nerve fibers of rat and guinea-pig pineal and in some pinealocytes of the latter. These findings speak in favour of the neuronal character of guinea-pig pinealocytes. The lack of neurofilament- and tubulin-immunoreactivity in rat pinealocytes might be attributable to very low concentrations of these proteins or species differences as to their expression. Further studies including in situ-hybridisation of relevant mRNAs will be necessary to answer these questions definitely.

Effect of low-molecular-weight factors of thymus and pineal gland on life span and spontaneous tumour development in female mice of different age.

Female SHR mice, aged 3.5 or 12 months, were exposed monthly to 5-day long courses of subcutaneous injections of 0.1 mg thymus-derived or pineal gland-derived polypeptide factors (TF and PF, respectively) or 0.9% sodium chloride solution (control). PF treatment increased life span of both young and middle-aged mice by 20% and 17%, respectively, and TF increased the life span only in young mice. Both factors when administered to young mice caused a decrease in both overall tumour incidence and incidence of mammary adenocarcinomas (TF, 1.8-fold decrease; PF, 2.6-fold decrease). TF administration to mature mice did not produce any antitumour effect, whereas PF possessed certain anti-tumour activity, but the response was far less pronounced than in young animals. The results obtained give additional evidence of the geroprotective and anti-tumour effect of thymus and pineal gland-derived peptide factors. The mechanisms of action of TF and PF and perspectives of clinical use of these agents as geroprotectors are discussed.

Effect of a pineal indolamine, 5-methoxyindole-3-acetic acid, on the estrous cycle and reproductive organs of female Wistar albino rats.

It has been suggested that the pineal gland has a specific role in the control of cyclic sexual activity in rats. One or more of the compounds isolated from this gland have been considered to be possible anti-fertility agents. In this study, the effect of different doses of 5-methoxyindole-3-acetic acid (5-MIAA) on the estrous cycle and the reproductive organs of female rats was studied. Daily injections of either 100 or 200 micrograms of 5-MIAA significantly prolonged the estrous cycles of mature female rats. After injections of 5-MIAA, an increase in diestrous smears was observed; in addition, the animals had estrous cycles without proestrous phase. Following the injections of 200 micrograms of 5-MIAA, a significant increase in uterine weight, plasma 17 beta-estradiol level and in the number of follicles were observed, and after injections of 5-MIAA (50, 100, 200 micrograms) a medium level of plasma progesterone was obtained. Thus it is concluded that 5-MIAA has an effect on the ovulation process of female rats.

Histochemical and ultrastructural study of the human pineal gland in the course of aging.

A histochemical and ultrastructural study was carried out on the pineal gland of 2 day- to 86-year-old subjects. Specimens were obtained by stereotaxis in the course of neurosurgery; other specimens were autoptical pineal fragments. Histochemical and ultrastructural analyses showed lipofuscin pigments, frequently localized in the proximity of blood capillaries and of cytoplasmic microacervuli. The brain sand amount does not appear to be age-related because microacervuli are barely present or frequently absent in the oldest subjects. Its involvement in the secretory activity rather than in gland atrophy is also suggested. The close relationship between cytoplasmic microacervuli and intermediate filaments suggests a possible role of the cytoskeleton in the formation of the brain sand.

Effect of environmental temperature and photoperiod on the melatonin levels in the pineal, lateral eye, and plasma of the frog, Rana perezi: importance of ocular melatonin.

Day-night melatonin concentrations were studied in the pineal body, lateral eye, and plasma of the frog Rana perezi in animals maintained in February and July under long (18L:6D) or short (6L:18D) photoperiod and high (25 +/- 1 degree) or low (6 +/- 1 degree) temperature in order to evaluate the influence of these environmental factors. When frogs were kept under short photoperiod and low temperature in February, no melatonin rhythm was observed in the pineal, ocular tissue, and plasma. High temperature at this period of the year induced a day-night rhythm of melatonin levels in the lateral eye and plasma. In July, under long photoperiod and high temperature, animals showed pronounced rhythms of melatonin in the pineal, eye, and plasma. A decrease of environmental temperature in this season abolished the melatonin rhythm. When animals were maintained in August under high (25 +/- 1 degree) temperature and long (18L:6D) or short (6L:18D) photoperiod, the duration of high night time ocular melatonin levels was correlated to the length of the dark phase. In all experiments the high ocular melatonin concentrations and the close parallelism observed between ocular and circulating melatonin profiles suggest that in this species melatonin could be released from the eyes in the general circulation.

Immunocytochemical localization of serotonin in the pineal gland.

The localization of serotonin (5HT) in the rat pineal gland during daylight conditions was investigated by light and electron microscopy using a polyclonal antibody to 5HT and the immunocytochemical method of peroxidase-antiperoxidase (PAP). Thin varicose 5HT fibers were found distributed in the gland at light microscopy level. Ultrastructural observations revealed the presence of terminal endings containing 5HT and localized mainly in the perivascular spaces, labeled with dense diaminobenzidine (DAB) deposits.

Antigonadotropic activity of pineal gland of the Indian palm squirrel Funambulus pennanti.

Bioassay of the pineal extract of F. pennanti was performed in immature female mice which was previously sensitized with human chorionic gonadotrophin. Reduction of ovarian and uterine weights indicated an antigonadotropic nature of the pineal gland of this animal.

Effect of psychotropic drugs on the contents of melatonin, serotonin and N-acetylserotonin in rat pineal gland.

In the dark phase, the effects of the psychotropic drugs on the contents of melatonin, serotonin (5-HT) and N-acetylserotonin (NAS) in rat pineal gland were examined. The pineal gland was removed at a certain period of time after subcutaneous injection of the drugs. 5-HT, NAS and melatonin contents in the pineal gland were determined by high performance liquid chromatography with fluorometric detection. A dose-dependent decrease was observed for melatonin content in the administration of diazepam (DZP), hydroxyzine (HYZ), chlorpromazine (CPZ) or haloperidol (HPD). When imipramine (IPM) or amitriptyline (APL) was given to rats, pineal 5-HT content was significantly decreased. On the other hand, the administration of IPM or APL caused increases in pineal NAS and melatonin. Furthermore, the administration of phenytoin (PYT) revealed no changes in the content of pineal indoleamines. These results suggest that the psychotropic drugs widely used in clinical applications could cause significant changes in pineal indoleamine content.

Predominant cytosolic distribution of serotonin in rat pineal gland in contrast to biogenic monoamine localization in midbrain and adrenal gland.

The subcellular distribution of serotonin and norepinephrine in the rat pineal gland was studied by tissue fractionation and compared with that of biogenic monoamines in the adrenal gland and midbrain. Homogenized tissues were fractionated by ultracentrifugation or by filtration through cellulose ester membranes. Most of the epinephrine (70-80%) and norepinephrine (62-82%) present in the adrenal glands was detected in the particulate fraction. The same distribution was found for serotonin (68.5%) and norepinephrine (59%) in the midbrain and for norepinephrine (62.5%) in the pineal gland. However, most of the serotonin in the pineal was found in the soluble fraction (89.5-98%). This suggests that the great majority of serotonin in the rat pinealocytes is cytosolic and thus is not stored in subcellular vesicles, in contrast to the biogenic monoamines in the midbrain or adrenal gland.

Partial purification of a polypeptide extract derived from ovine pineal that suppresses the growth of human melanoma cells in vitro.

A combination of gelfiltration and reverse-phase high performance liquid chromatography with postcolumn antitumour assay has been developed. A melatonin insensitive human melanoma cell strain was used to guide the purification of the antitumour effect of an ovine pineal aqueous extract (MW 1,000 to 10,000) that possessed the ability to decrease the hypophysiotropic activity of rat and mice hypothalami in vitro. This allows a specific identification of a pineal factor (MW 2,000 to 6,000) that inhibits the growth of human melanoma cells at a dose of 0.47 mg/ml medium. It was shown that the activity of this pineal compound differs from structures known to be present in the pineal, such as melatonin, pteridines, and beta-carbolines. There appears to be evidence for a peptidic nature of this pineal antitumour factor.

Serotonin and melatonin contents in the pineal glands from different stocks and strains of laboratory rats.

In the present study the pineal gland was examined in 2 outbred stocks and 6 inbred strains of rats some of which were pigmented to varying degrees, to see whether inbreeding affects the variability and whether differences exist between albino and pigmented rats. The animals were kept under 12 h light: 12 h darkness (12 L:12 D) and killed 7 h after the onset of light and darkness, respectively. The parameters examined were pineal protein content, serotonin and melatonin levels and hydroxyindole-O-methyltransferase (HIOMT) activity. All the parameters examined revealed interstrain differences, independently of whether the data were expressed per pineal or per mg protein. The variation coefficients for the various parameters were relatively high. They were mostly smaller when the data were expressed per pineal rather than per mg protein. No striking differences existed between the variation coefficients in inbred and outbred rats. When pineal size and the melatonin-related parameters expressed per pineal were used to assess the melatonin-synthesizing capacity of the pineal glands, it was found that the outbred Wistar and Sprague-Dawley rats and the inbred LEWIS-derived (LEW/Han) rats, all of which were albinos, had the most active pineals. Intermediate activity was noted in the hooded E3/Han and BDE/Han and the albino BDII/Han rats. The smallest and least active pineals were found in the totally pigmented BN/Han and DA/Han rats. The results taken together show that different stocks and strains exhibit significant differences in pineal size and melatonin-forming capacity. Albino rats appear to have larger and more active pineals than pigmented rats.

Influences of the paraventricular and suprachiasmatic nuclei and olfactory bulbs on melatonin responses in the golden hamster.

Removal of the pineal, or denervation of this gland by superior cervical ganglionectomy, blocks testicular regression in golden hamsters exposed to short photoperiods. Aspiration of the olfactory bulbs or lesions of the suprachiasmatic or paraventricular nuclei of the hypothalamus (SCNx or PVNx) have similar effects. We have examined the effects of these operations on pineal melatonin content and gonadal responses to various patterns of exogenous melatonin in order to examine the roles played by the olfactory bulbs, the SCN, and the PVN in hamster photoperiodism. SCNx and PVNx significantly reduced pineal melatonin content throughout the dark phase, while bulbectomy did not significantly affect melatonin concentrations at the time of the nocturnal peak. Bulbectomy significantly delayed the nightly onset of locomotor activity in hamsters exposed to 14L:10D, but not that of animals housed in 10L:14D. Although bulbectomy reduced the gonadal response to one or three daily injections of melatonin, these individuals exhibited significant testicular regression in response to melatonin as long as injections fell in the evening. In contrast, destruction of the PVN rendered hamsters unresponsive to one daily melatonin injection, but equally responsive to three injections, regardless of the time of day at which these injections were given. Whereas exposure of bulbectomized hamsters to 30 weeks of short days made them refractory to subsequent melatonin challenge, PVNx hamsters remained sensitive to appropriately timed melatonin treatments regardless of their photoperiodic history. Many, but not all hamsters that experienced complete SCN lesions remained sensitive to three daily melatonin injections.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural study of the distribution of calcium in the pineal gland of the rat subjected to manipulation of the photoperiod.

Using the pyroantimoniate technique, a study was conducted at electron microscope level on the distribution of the calcium ion in the pineal glands of normal adult Sprague-Dawley rats with initial weights of 150-200 g subjected to a 12:12 light dark cycle and others under the same conditions were subjected to modifications in the noradrenergic signal, such as continuous illumination over 7 days, blinding by bilateral enucleation (7 or 90 days) before sacrifice and bilateral superior cervical gangliectomy at 21 days before sacrifice. All the animals were sacrificed by decapitation, half of them at midday and the other half at midnight. Abundant fine precipitations of calcium were found in the intercellular spaces of the pineal glands of the normal rats. By contrast, in the gangliectomized animals subjected to constant illumination and chronic binding these precipitations were few in number. Additionally, two types of pinealocytes were observed regarding the distribution and concentration of intracytoplasmic calcium in both the normal and experimentally manipulated animals. Type I correspond to the classic light pinealocytes, with an absence of intracytoplasmic precipitations, although in the normal and gangliectomized animals sacrificed at midnight it was possible to observe fine deposits inside the mitochondrial matrix. Type II correspond to the classic dark pinealocytes, with a dense cytoplasmic matrix and numerous deposits of intracytoplasmic and intranuclear calcium; these were never seen in the type I pinealocytes.

Immunoelectron microscopy of rhodopsin and vitamin A in the pineal organ and lateral eye of the lamprey.

Rhodopsin- and vitamin A-immunoreactive sites were studied in the pineal organ of the larval and adult brook lamprey (Lampetra planeri Bloch), as well as in the retina of the larval lateral eye, at the electron microscopic level. In the pineal organ, several types of photoreceptor cells could be distinguished by their morphology and the immunoreactivity of their outer segments. The different kinds of photoreceptor cells were located at different levels of the pineal organ according to their distance from the "pineal window". The most superficial level, the so-called pellucida, appears to represent an exclusively "cone-type" area containing slender, rhodopsin-immunonegative (UV-blue-sensitive?) photoreceptors only. The second level, the pineal retina, contained predominantly rod-type photoreceptors, i.e., large, strongly rhodopsin-immunopositive (green-sensitive) photoreceptors medially, and few, small, weakly rhodopsin-immunopositive (blue-green-sensitive?) cells bilaterally. At the deepest level, the pineal atrium, there were both rod- and cone-type photoreceptor cells, the latter possibly representing red-sensitive elements. Vitamin A immunoreactivity was found in the outer segments of the pineal photoreceptor cells, in the cytoplasm and mitochondria of inner segments and perikarya, as well as in nuclear euchromatin and compact nucleoli. A similar gold labelling of organelles was observed in the ependyma and pineal neurons. The vitamin A immunoreaction of the outer segments suggests retinoids are present as chromophores of the photopigments. In the peripheral retina of the larval lateral eye, vitamin A immunoreactivity was found in some organelles of the undifferentiated photoreceptor cells, neurons, pigment epithelium and Müllerian cells. The localization of immunoreactive vitamin A in nuclei, nucleoli and cytoplasm including mitochondria appears to strengthen the case for an interaction of retinoids in the function of these organelles.