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.     

Characterization of a Neurohypophyseal Hormone-Like Activity Isolated From Ovine Pineal Glands

The milk-ejecting response of lactating mouse mammary gland tissue to ovine pineal extracts indicated the presence of a neurohormone-like bioactivity in this tissue. After successive fractionation on gel permeation chromatography and reversed-phase liquid chromatography (HPLC) in conjunction with radioimmunoassays (RIA), it was demonstrated that the milk-ejection response to ovine pineal components with an Mr less than 1,000 corresponded to a biologically active peptide sequence that probably differs from that of arginine vasopressin, arginine vasotocin, and oxytocin and from peptides with a COOH-terminal Pro-Arg-Gly-amide ending. Gel permeation chromatography in formic acid appeared also to indicate the presence of a noncovalent interaction of the neurohormone-like bioactivity with proteins (Mr greater than 25,000) of the pineal.     

Evidence for Dual Adrenergic Receptor Regulation of Ovine Pineal Function

Both alpha 1- and beta-adrenergic receptors are present on ovine pineals. In the rat these two receptors interact so that activation of the alpha 1-receptor potentiates the beta-receptor-mediated changes in cyclic AMP and the correlated changes in pineal N-acetyltransferase (EC 2.3.1.87). Here we investigate possible interactions between alpha 1- and beta-receptors through changes in cyclic AMP and assess the importance of each receptor to the melatonin response in ovine pineal punches/slices in vitro. The adrenergic agonists isoproterenol (ISO), noradrenaline (NA), and phenylephrine (PHE) stimulated dose-dependent changes in cyclic AMP with the order of potency of ISO greater than NA greater than PHE, consistent with their relative binding affinities for the beta-receptor. The beta-receptor antagonist, propranolol, showed dose-dependent inhibition of the ISO effect, whereas the alpha 1-selective antagonist, prazosin, had no effect. The S-shape of the stimulation and inhibition curves for ISO reflects cyclic AMP changes mediated by the beta-receptor only without interaction through the alpha 1-receptor. Each of the adrenergic agonists stimulated indistinguishable dose-dependent increases in melatonin release. The ability of PHE to stimulate changes in melatonin release in the absence of concomitant changes in cyclic AMP indicates an important role for the alpha 1-receptor. Prazosin inhibits this response, substantiating this conclusion. However, as propranolol is also inhibitory, it seems that the alpha 1-receptor response is absolutely dependent upon a small level of beta-receptor stimulation, thus providing evidence for adrenergic receptor interaction at a step other than cyclic AMP.     

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.     

Rhythms in Pineal Immunoreactive Somatostatin in the Syrian Hamster, Mouse, and Gerbil

Immunoreactive somatostatin (IRS) has been previously demonstrated in the pineal gland of different rodent species, and we observed a 24-hr rhythm in rats. Recent data suggest that the peptide may represent a neurotransmitter in the so-called peptidergic nerves of the central, pinealopetal innervation of the epiphysis, which may modulate the activity and secretion of the gland. We investigated whether 24-hr changes of pineal IRS content occurred in Syrian hamsters, gerbils, and mice. Adult males, kept in a 14:10 LD photoperiod, were decapitated at 4-hr intervals throughout a 24-hr period. Pineals and median eminences were analyzed for IRS by radioimmunoassay. No significant changes in the median eminence content of IRS with time was observed. As previously described in rats, a statistically significant rhythm of IRS was observed in the pineal of hamsters and mice, with a peak at 2000 hr (mice 51.7 +/- 5 pg/pineal; hamsters 26.3 +/- 4.6) and a nadir at 2400 hr (mice 30.8 +/- 1.4) or 0400 hr (hamsters 8.6 +/- 1). However, in the gerbil pineal IRS content remained unchanged throughout the period of study. Since the three species examined have very different melatonin cycles, it is suggested that the melatonin and IRS rhythms are unrelated and independently regulated events within the pineal gland.     

No Correlation of Pineal "Synaptic" Ribbon Numbers and Melatonin Formation in Individual Rat Pineal Glands

As previous circadian studies of pineal "synaptic" ribbon numbers and melatonin formation suggested that a positive correlation of the two variables exists, in the present investigation this problem was examined in individual pineal glands of rats killed at 1200 h and 2400 h, respectively. For this purpose, one half of the gland was processed for electron microscopy and the ribbons were counted in an area of 20,000 micron2 tissue; in the other half serotonin N-acetyltransferase (NAT) activity and melatonin content were determined. No correlation was found to exist between ribbon numbers and pineal NAT activity, pineal melatonin levels and serum melatonin levels, either at day- or at nighttime. It is concluded that the ribbons may perhaps be more closely related to the innervation of the pineal gland than to melatonin formation.     

Daily Profiles of N-Acetyltransferase Measured at a Single Time in Rat Pineal Glands, Retinas, and Harderian Glands

Serotonin N-acetyltransferase activity (NAT) exhibited a daily cycle in light:dark (LD) 14:10 when it was measured in pineal glands taken from rats killed at a sequence of time points. The ratio of peak subjective night NAT to minimum subjective day NAT was 10.9/0.3 nmol per pineal gland per hour. When the rats were placed in constant dark the rhythm persisted (8.2/0.02). When the rats were placed in constant light the rhythm persisted with markedly attenuated amplitude (0.6/0.02).
We also measured NAT profiles in rat pineal glands, Harderian glands, and retinas with alternative methods. We kept rats on six LD 14:10 light-dark cycles with lights-out beginning at midnight, 2 AM, 4 AM, 6 AM, 8 AM, or 10 AM and killing all the rats at one time point, 10 AM. We examined the NAT time profiles 4, 8, and 11 days following placement of the rats in the phase-shifted cycles. In addition, we measured the NAT profile in LD 2:22 and LD 22:2 by keeping the rats on twelve cycles for 11 days and killing all the rats at one time.
Pineal NAT exhibited a rhythm in all the cycles: peak-dark/nadir-light values (nmol product per gland per hour) were 15.6/0.1 in LD 14:10, controls killed at successive time points. The ratios for the profiles obtained using the one time point procedure were 16.7/0.1 in LD 14:10 8.5/0.2 in LD 22.2, and 12.9/0.2 in LD 2.22. Increasing the photoperiod reduced the time to the NAT peak.
In LD 14:10, Harderian NAT was 31–39 nmol per gland per hour but the peak/ nadir radio was only 1.2; retinal NAT was low (0.2–0.7 nmol per retina per hour) and had only a 3.5-fold peak/nadir ratio.     

Characterization of Dopaminergic Receptor Sites in Bovine Pineal Gland

In addition to beta-adrenergic receptor agonists, L-dopa and dopamine have been also shown to activate the production of melatonin and its synthesizing enzyme, serotonin N-acetyltransferase. In an attempt to characterize dopaminergic receptor sites, bovine pineal synaptosomes were prepared by differential centrifugation techniques. Washed disrupted synaptic membranes were used to study 3H-spiroperidol binding, using standard membrane-binding techniques. Association of 3H-spiroperidol to pineal membranes was very rapid, reaching equilibrium within 2 min and remaining stable for 20 min. Dissociation was also rapid with a t 1/2 of 3 min. Analysis of saturation studies (0.035 to 20 nM spiroperidol, 16 concentrations) using the LIGAND program indicated the presence of two binding sites with KDS (dissociation equilibrium constant) of 0.18 nM and 2.1 nM. The Bmax's (receptor density) of the sites were 37 and 630 fmoles/mg protein respectively. The IC50S of haloperidol, cis-flupenthixol, and chlorpromazine were 8, 12, and 80 nM, respectively, while those of pipamperone, cyproheptadine, and cinanserin were 60, 400, and 1500 nM. These and other data indicate that the most abundant site is a dopamine D2 receptor while the less abundant site may be a serotonin receptor. The function of dopamine and dopaminergic neurons in bovine pineal gland is not known and has not been established.     

Production of Methoxyindoles In Vitro From Methoxytryptophan by Rat Pineal Gland

Pineal glands were incubated in the presence of [3H] methoxytryptophan with and without methoxamine, epinephrine, and norepinephrine. The beta-adrenoceptor-stimulated pineal glands were capable of converting methoxytryptophan to methoxytryptamine, melatonin, methoxyindole acetic acid, and methoxytryptophol, albeit in small quantities. Only methoxyindole acetic acid was detectable after incubation of unstimulated and alpha-adrenergic-agonist-treated pineal glands. These results support the proposal that melatonin can be formed from methoxytryptophan although this is a minor synthetic pathway, and the classic pathway from serotonin via N-acetylserotonin should be considered to be responsible for the majority of pineal melatonin production.     

The Effect of Copper on (3H)-Tryptophan Metabolism in Organ Cultures of Rat Pineal Glands

Copper toxicity has been implicated in various neurodegenerative disorders such as Wilson's disease and Alzheimer's disease. Free copper in the brain is toxic and leads to neuronal and cellular damage, through free radical generation. Melatonin has been investigated as a possible copper ion chelator. Melatonin could prevent copper-induced neuronal and cellular damage through binding with copper and preventing copper-induced free radical generation. The effect of copper on pineal indolamine synthesis has not been studied extensively. In the present study, copper (2 mg/kg) and melatonin (12 mg/kg) were administered daily to Wistar rats for a 2-week and 6-week period. Pineal organ culture was utilized to monitor pineal indolamine synthesis. The pineals from the 2-week copper/melatonin-treated group showed a statistically significant decrease in 5-methoxytryptophol synthesis (p < 0.01), compared to the pineals from the copper-treated group. Conversly, in the 6-week experiment, 5-methoxytryptophol synthesis was increased in both the copper- and copper/melatonin-treated groups. There was a statistically significant decrease in the N-acetyl serotonin level in the pineals from the 6-week copper-treated animals, as compared to the control- and copper/melatonin-treated group (p < 0.01). These results imply that copper reduces N-acetyltransferase activity, which results in a decrease in N-acetyl serotonin synthesis. Melatonin when coadministered with copper appears to prevent the N-acetyltransferase inhibition by copper. Copper exerts contradictory effects on 5-methoxytryptophol synthesis. Further investigations need to be carried out to examine the effects of copper on the pineal enzymes.     

Interdependent Effects of the Ionophore A23187 and Serum on the Serotonin N-Acetyltransferase Activity of Cultured Chick Pineal Glands

Marked effects of the ionophore A23187 on the cycle of N-acetyltransferase (NAT) activity in cultured chick pineal glands were observed under three conditions of illumination. However, the effects were qualitatively and quantitatively dependent on the batch of fetal calf serum used in the medium and time of explanation into culture. Ionophore increased the level of NAT activity remaining in glands exposed prematurely to light regardless of the serum used. The ionophore suppressed the "spike" in cyclic GMP content of glands cultured in the dark, and extended the period of maximum cyclic GMP content of glands under diurnal illumination.     

Interaction of arginine vasotocin and norepinephrine upon pineal indoleamine synthesis in vitro

The interaction of arginine vasotocin (AVT) and norepinephrine (NE) upon pineal gland indoleamine synthesis was investigated. Rat pineal glands were incubated for 10 h in Krebs-Ringer bicarbonate plus 2 mg/ml glucose, 1 mg/ml bovine serum albumin, [¹⁴C]tryptophan, NE (10^?6 M), and log doses of AVT ranging from 100 ng to 10 μg. Incubation media were extracted for [¹⁴C] serotonin while the other [¹⁴C]indoleamines, melatonin, hydroxyindoleacetic acid (HIAA), methoxyindoleacetic acid (MIAA), N-acetylserotonin (NAS), hydroxytryptophol (HTOL), and methoxytryptophol (MTOH) were separated by thin-layer chromatography. Serotonin metabolism was decreased by 0.1 μg AVT and NAS decreased by 1.0 μg AVT. Melatonin synthesis was decreased by both 0.1 and 1.0 μg AVT. AVT also decreased the conversion of [¹⁴C]serotonin to MIAA and to HTOL. The data indicate that AVT decreased NE-stimulated pineal indoleamine synthesis in vitro and further suggests that AVT may participate in the intracellular regulation of melatonin synthesis.     

Alloxan-Induced Diabetes and the Pineal Gland: Differential Effects on the Levels of Pineal N-Acetylserotonin, Pineal Melatonin, and Serum Melatonin

Effects of alloxan treatment on the levels of pineal melatonin, pineal N-acetylserotonin, and serum melatonin were investigated. Male rats were housed under a photoperiod of 12 h light: 12 h darkness and a temperature of 23 +/- 3 degrees C. Three weeks after alloxan (170 mg/kg) or carrier injection (s.c.), the animals were killed at mid-light (1200 h) and mid-dark (2400 h). Pineal and serum indoles were extracted and quantified by radioimmunoassays. It was found that pineal levels of N-acetylserotonin in the diabetic rats were significantly higher (P less than 0.05) than those of the controls. Conversely, pineal and serum levels of melatonin in the control rats were significantly higher (P less than 0.05) than those of the alloxan-induced diabetics. Our results suggest that alloxan-induced diabetes may decrease pineal melatonin synthesis in rats by reducing the activity of hydroxyindole-O-methyltransferase, resulting in a decrease in pineal melatonin secretion.     

The Ultrastructure of the Nerve Fibers and Pinealocytes in the Rat Pineal Stalk

In view of the increasing interest in the central innervation of the mammalian pineal gland, this aspect was studied in depth in the rat. This species is especially suited since the nerve fibers in question form a distinct bundle running from the deep to the superficial pineal gland through the pineal stalk. The axons were counted and analysed ultrastructurally in the pineal stalks cut transversely at three levels (proximal, intermediate, and distal) relative to the neural axis and in longitudinal sections. The number of nerve fibers was highly variable, ranging from 551 to 1,132 proximally and from 110 to 448 distally, indicating that many fibers terminate in the stalk or leave the stalk after forming a loop. Large myelinated axons, which are abundant proximally, appear to lose their sheaths along their course through the stalk. Most of the axons were small and unmyelinated. A few of these had the appearance of sympathetic fibers and disappeared after sympathectomy. Others contained abundant neurosecretory granules, and, according to the literature, may originate in the hypothalamic paraventricular nuclei. The majority of the small axons which are apparently devoid of granules and dense-cored vesicles may come from the habenular nuclei and the stria medullaris. In addition to axons, the stalk contains astrocytes, a few oligodendrocytes and Schwann cells, as well as pinealocytes identical to those of the superficial pineal gland.     

Pineal modulation of the rat caudate-putamen spontaneous neuronal activity: Roles of melatonin and vasotocin

Abstract: The effects of microiontophoretic application of melatonin and melatonin plus vasotocin on spontaneously active neurons of caudate-putamen in sham-operated and pinealectomized rats were studied. Extracellular unit recordings showed that in sham-pinealectomized rats, melatonin ejection primarily produced inhibition of the responsive neurons (74.1%), whereas only 24.9% of the neurons were excited. Iontophoretic ejection of vasotocin or melatonin + vasotocin produced, in both cases, an inhibition of 100% of the responsive neurons. In pinealectomized rats, iontophoretic melatonin ejection produced a similar percentage of inhibition (46.1%) and excitation (53.8%) of the responsive neurons. The simultaneous ejection of melatonin + vasotocin further increased the percentage of inhibition (88.8%) compared with the melatonin only treated group. Moreover, iontophoretic ejection of vasotocin inhibited 100% of the responsive neurons in pinealectomized rats. The actions of melatonin and vasotocin seem to be specific, because their effects are dependent on the amount of these compounds ejected, i.e., the intensity of the ejection current. These results indicate that the pineal compounds melatonin and vasotocin are neuromodulators of spontaneous neuronal activity of the rat caudate-putamen.     

Circadian Rhythms of Human Pineal Melatonin, Related Indoles, and Beta Adrenoreceptors: Post-Mortem Evaluation

Human pineal glands obtained from 77 post-mortem sources from various age groups and times of death were used to examine the 24-hour cycle of serotonin (5-HT), melatonin, N-acetylserotonin (NAS), and beta adrenoceptor density. Pineal glands were divided sagitally and a single half was used to measure 5-HT, NAS, and melatonin concentrations, while the remaining half from the same gland was employed to assess changes in the density of beta adrenoceptors on partially purified membranes. The results show that density of pineal beta adrenoceptors was relatively constant between midnight and 18.00 h and became significantly higher between 18.00 and 20.00 h as measured by ligand saturation binding experiments using (125-1) iodocyanopindolol. The receptor affinity of all of the samples assayed remained in relatively narrow range near 58 pM and only changes in the relative receptor density were apparent. The up-regulation of receptors coincided with an increase in the concentration of 5-HT that began to rise between 16.00 and 20.00 h and became maximal between 20.00 and midnight. NAS, the immediate precursor of melatonin, was also at maximal levels between 20.00 h and midnight. Both 5-HT and NAS began declining after midnight and this change corresponded to the maximal pineal gland concentration of melatonin between midnight and 4.00 h. It is therefore suggested that the up-regulation of beta adrenoceptors noted during the late afternoon and early evening hours corresponds to the increased synthesis of 5-HT and the subsequent conversion to NAS. These events are followed by the highest accumulation of melatonin after midnight and represent the synthesis of melatonin from its precursor NAS in a sequential pattern.     

Effects of Melatonin and Control Injections on Pineal Serotonin and Norepinephrine: Afternoon Injections Lower Serotonin Levels Thirty-Six Percent at Light-Dark Transition

Pineal weight and serotonin (5-HT) and norepinephrine (NE) contents were studied in male Sprague-Dawley rats that were maintained under controlled light:dark conditions (LD 14:10; lights on 0700-2100) and that received daily subcutaneous injections of either melatonin (20 μg in 0.1 ml per animal) or the same volume (0.1 ml) of vehicle alone, at one of two times (0800-0900 or 1800-1900). Animals were sacrificied at four times (1000, 1400, 2000, or 2300) on the day after the last of the 7 consecutive d of injection. Pineal glands were quickly weighed and then frozen for 5-HT and NE assay by the Maickel and Miller extraction and fluorescence methods.
Pineal NE content showed differences related to time of day, in confirmation of early work. But no effects attributable specifically to melatonin were found. Melatonin also failed to affect pineal 5-HT content significantly. But injection of either melatonin or vehicle at 1800-1900 led to a reduction in 5-HT content averaging 36% when sampled at either 2000 or 2300, and in comparison with animals injected at 0800-0900. It is suggested that a stresslike or zeitgeberlike effect of injections within a critical period at the end of the daily light phase can cause an earlier-than-normal daily fall in pineal 5-HT content.     

Age-Related Incidence of Pineal Gland Calcification in Children: A Roentgenological Study of 1,044 Skull Films and a Review of the Literature

Anterior-posterior and lateral skull roentgenograms of 1,044 children aged 0-18 yr were examined for pineal gland calcification. Eighty children with pineal calcification were identified. Cranial computed tomograms (CCT) existing for half of the 80 cases provided confirmation. In contrast to existing reports on pineal calcification in the first decade of life, we found a significant percentage of "physiological" calcification even between 0 and 6 yr of age (range 2.9-4.2%). Contrary to current opinion we were not able to detect any signs of pineal gland tumors in these cases. We were able to confirm other reports which note a steep rise of the incidence of pineal calcification during the second decade of life.     

Differential Effect of Carbidopa on the Concentration of Rat Pineal and Hypothalamic Indoleamines

Carbidopa, an aromatic acid decarboxylase inhibitor, has been shown to significantly decrease the pineal concentration of melatonin, N-acetyl serotonin (NAS), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) but not the hypothalamic concentrations of these indoles. Increased levels of 5-hydroxytryptophan (5-HTP) indicate that carbidopa directly inhibits 5-HTP decarboxylation, thus limiting 5-HT production. Possible practical implications of selective inhibition of pineal indoleamines by carbidopa are discussed.     

Type II Thyroxine 5''-Deiodinase Activity in the Rat Brown Adipose Tissue, Pineal Gland, Harderian Gland, and Cerebral Cortex: Effect of Acute Cold Exposure and Lack of Relationship to Pineal Melatonin Synthesis

The effect of acute cold exposure for 6 hours on nocturnal type II thyroxine 5'-deiodinase (5'-D) activity was studied in brown adipose tissue (BAT), Harderian gland, cerebral cortex, and pineal gland of the rat. Moreover, the effect of iopanoic acid (IOP), a potent inhibitor of 5'-D activity, on both pineal N-acetyltransferase (NAT) activity and melatonin content in rats maintained in a cold environment was also examined. Results show that acute cold exposure significantly increases 5'-D activity in BAT but not in either the pineal gland, Harderian gland, or cerebral cortex. In all tissues, the injection of IOP reduced dramatically 5'-D activity, while exposure of the animals to light at night reduced 5'-D activity in pineal gland but not in either the Harderian gland or BAT while light exposure at night increased cerebrocortical 5'-D activity. Cold exposure did not change either pineal NAT activity or the melatonin content of the gland. Finally, when pineal 5'-D activity was inhibited by IOP treatment, neither nocturnal pineal NAT activity nor melatonin content was affected.