Structure of a pineal gland-derived antigonadotropic decapeptide.

An antigonadotropic decapeptide was extracted from bovine pineal glands, purified by ultrafiltration and Sephadex G-25 gel-filtration chromatography and isolated by serial semi-preparative high performance liquid chromatography. Its primary structure was determined by automated amino acid and microsequence analyses. A synthetic analog of the decapeptide, prepared by solid state methods, was observed to reduce circulating levels of luteinizing hormone and prolactin after intra-atrial injection in male rats.

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland.

The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M(r) range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [125I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (Ka of 4.7 +/- 0.2 x 10(9) M-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M(r) 24,000. The functional status of PRL- and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.

Identification of luteinizing hormone-like proteins in the ovine pineal gland.

A chemical analysis was instigated to investigate the identity of the luteinizing hormone (LH)-like immunoactivity present in ovine pineal protein homogenates. Isolation of pineal LH-like material was accomplished using a 0.1 M ammonium sulphate (pH 4.0) extraction followed by anion-exchange chromatography. The resulting 3.0 M ammonium sulphate precipitate containing 70% of the LH-like immunoactivity was refractionated by cation-exchange and Sephadex G-100 chromatography. Analysis of the pattern of recovered LH-like immunoactivity in the Sephadex G-100 eluate indicated the presence of molecular weight (MW) less than 60,000 besides MW 21,000 species of LH-like proteins. Bioactivity was tested in the rat Leydig cell steroidogenesis assay. In terms of steroid production, the activity was associated with the MW 21,000 LH-like proteins only. Further purification by CM-Sephadex chromatography and gel permeation HPLC was conducted in order to determine whether the physicochemical properties of pineal LH-like material represented endogenous LH, synthesized and released by the ovine pituitary. It is concluded by a variety of means, including polyacrylamide gel electrophoresis, and amino acid and carbohydrate analyses, that at least two molecular forms of immunoactive LH-like proteins occur in ovine pineal tissue. The MW 21,000 forms showed much similarity with ovine adenohypophyseal LH or with a complex mixture of its subunits. These observations contribute to the understanding of endocrine-endocrine transducing events that may occur in this organ.

Antitumor activity of the pineal gland: effect of unidentified substances versus the effect of melatonin.

There is growing evidence that the pineal gland has antineoplastic properties which, however, can only partially be attributed to its hormone melatonin. While the in vivo tumor-inhibiting activity of melatonin is established, observations on its in vitro effects have been contradictory. The effect of this substance was investigated on six human cancer cell lines and compared to the activity of a partially purified, melatonin-free low molecular weight pineal extract (UMO5R). Melatonin showed hardly any effect but UMO5R was capable of inhibiting the growth of all the six cell lines tested. It is therefore concluded that a direct inhibiting action on tumor cells is not a general physiological role of melatonin as opposed to UMO5R. It will be worthwhile to purify the yet unidentified pineal antitumor activity since it may have a considerable therapeutic potential.

Isolation and structure elucidation of bovine pineal arginine vasopressin: arginine vasotocin not identified.

A large number of reports have demonstrated the presence of neurohypophysial hormone-like peptides in mammalian pineal glands and an antigonadotropic function has been ascribed to pineal arginine vasotocin (AVT). We have undertaken large scale purification of bovine pineal neurohypophysial hormone-like substances which demonstrate mouse mammary milk-ejection activity (ME-activity) in vitro. Peptides with ME-activity were extracted from more than 5 kg of bovine pineal glands. ME-activity containing peptides were found in both high (Mr approximately 10,000-15,000) and low (Mr approximately 500-1000) Mr species from Sephadex G-25 chromatography of 0.2 N acetic acid extracts. After ultrafiltration in 5% formic acid, the neurohypophysial hormone-like peptides were localized to an ultrafiltration Mr 500-1000 retentate. A homogeneous peptide, which shared an identical retention time (RT) and amino acid sequence with synthetic 8-arginine vasopressin (AVP), was isolated by serial semipreparative high performance liquid chromatography. On the other hand, the non-mammalian nonapeptide AVT was not identified.

The influence of light of different wavelengths on the methylating capacity of the pineal gland of male golden hamsters in relation to reproduction.

In the present experiments the influence of light of different wavelengths on pineal indole metabolism in relation to reproduction was studied. Therefore, during autumn and winter male golden hamsters were kept under natural conditions but for the sunlight which was filtered exposing the hamsters to either normal (control), red or blue light. During the gradually shortening photoperiod at the start of the experiments under normal light conditions, a marked decrease of FSH and LH plasma content as well as testicular weight was found, indicating the onset of gonadal atrophy. During this period a high synthesis of 5-methoxytryptophan (MW) and 5-methoxytryptamine (MT) was determined. The synthesis of other 5-methoxyindoles (MI) was low, while O-acetyl-5-methoxytryptophol (aML) synthesis even markedly decreased. Red and blue light did not cause significant changes in MI synthesis. As long as MT synthesis is high (under blue light), there is no increase in FSH content and testes weight is still decreasing. This influence of blue light confirms the putative antigonadotropic properties of MT. The increase of FSH content at week 9 was the first indication that recrudescence had started. At week 19, this recrudescence was also manifested in the increasing testes weight. The synthesis of melatonin (aMT), 5-methoxytryptophol (ML), 5-methoxyindole-3-acetic acid (MA) and aML increased whereas the production of MT decreased. Blue light exposure caused a significantly higher increase of synthesis of ML, MA, aML and, not-significantly, of aMT, whereas red light caused a significantly lower synthesis of MA. It was concluded that MT, a putative antigonadotropic, and aML, a putative counter-antigonadotropic, are probably important pineal compounds that transduce the photoperiodic messages, which cause either gonadal atrophy or recrudescence. The effect of blue light on indole metabolism and the reproductive cycle was more clear than that of red light. From the present results of blue light on indole metabolism, it was suggested that blue light delayed gonadal atrophy and stimulated gonadal growth, compared to red light. An opposite effect of red light was less obvious.

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.

Neurohypophyseal hormone-like peptides in the ovine pineal gland using reverse-phase liquid chromatography and radioimmunoassay.

A method is described for the determination of the neurohormone contents of ovine pineal tissue by radioimmunoassay (RIA) after successive fractionation on gel filtration in formic acid and reverse-phase liquid chromatography (HPLC). This method gives a good resolution for the neurohormones vasopressin, vasotocin and oxytocin, without a significant interference of aspecific cross-reacting of peptides with the RIA. An acid extract from ovine pineal tissue was found to contain amounts of immunoreactive AVP- and OXT-like peptides, whereas an AVT-like peptide was not detectable over background levels after HPLC with post-column RIA. It is concluded from our results that an AVT-like peptide is not present in ovine pineal tissue, and the pineal AVP- and OXT-like peptides appeared to be associated to neurophysin molecules.

Partial purification of (a) low molecular weight ovine pineal compound(s) with an inhibiting effect on the growth of human melanoma cells in vitro.

An in vitro human melanoma cell assay was used to work up the partial purification of (a) low molecular weight (MW) substance(s) from aqueous extracts of ovine pineal tissue shown to contain a growth-inhibiting activity. A combination of paper chromatography, ion-exchange and reverse-phase high performance liquid chromatography with post-column antitumor assay has been developed. This allows a specific identification of an ovine pineal factor (MW less than 500) which inhibits the growth of human melanoma cells in vitro. The substance was partially purified to about 1,000 times as compared to the IC100-value of the starting material (retentate 5). The growth inhibition of human melanoma cells in culture was complete at a dose of 0.1 microgram/ml of purified pineal factor(s). It was demonstrated that the activity of this pineal compound differs from some substances known to be present in the pineal, such as melatonin, serotonin, peridines and beta-carbolines. The activity was not destroyed by treatment with proteolytic enzymes.

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.

Purification and characterization of DSIP-like material from ovine pineal glands: possible peptide-protein interaction.

The nonapeptide delta-sleep-inducing peptide (DSIP) has been isolated from venous blood of rabbits induced to sleep. Numerous reports have described sleep as well as extra-sleep effects. Radiochemical and immunochemical data suggest a relationship of DSIP with the pineal gland supported by interactions of this peptide with pineal functions such as the serotonin N-acetyltransferase activity. In order to demonstrate the natural occurrence of DSIP-like material associated with high Mr proteins in the ovine pineal, organs were water-extracted and fractionated by ultrafiltration and gel filtration. Radioimmunoassay (RIA) for DSIP-like fragments of the fractions revealed considerable amounts of pineal DSIP-like immunoreactivity (DSIP-LI) apparently existing in small as well as large molecular forms. Acidification of large DSIP-LI forms resulted in the elution from Sephadex G-50 of Mr less than or equal to 1,000 DSIP-like material. This free DSIP-LI form coeluted with the synthetic DSIP nonapeptide from microBondapak C18 on high-performance liquid chromatography. The results, therefore, appear to indicate the presence of a (biospecific) noncovalent intermolecular interaction of DSIP (1-9) with proteins (Mr greater than or equal to 10,000) of the ovine pineal gland.

Studies on a high molecular weight luteinizing hormone release stimulating factor of the ovine pineal gland.

Former work has shown that crude extracts of ovine pineal glands probably exert a stimulating activity on the release of gonadotropins of anterior pituitaries in vitro. By aqueous extraction followed by ultrafiltration through anisotropic membranes high Mr (above 100,000 daltons) fractions were obtained, which exhibit a stimulating effect on the levels of gonadotropins in the medium of either cultured pituitary cells or anterior hemipituitaries in short-term culture. Partial purification of a pineal luteinizing hormone release stimulating factor was accomplished by Sephadex G-150 filtration with a biopotency of 226 +/- 23 micrograms LH-RP-1 equivalents per mg protein and without an affinity for binding to anti-LHRH or anti-LH antibodies. The present data substantiate that high Mr forms, slightly heavier than authentic pituitary LH (Mr 23,000 daltons) and therefore not identical to the hypothalamic decapeptide LH-RH, represent ovine pineal factors which can increase the concentration of LH in the medium of cultured anterior pituitaries, but does not influence the secretion of prolactin in vitro.

Modified forms of vasopressin and oxytocin in a bovine pineal preparation.

A bovine pineal acid extract displays a vasotocin-like bioactivity in several bioassays, and is recognized by antibodies against the Pro-Arg-Gly-amide ending common to vasopressin and vasotocin. By using molecular sieve filtration and reversed-phase HPLC, a vasopressin- and oxytocin-like peptide was isolated from this pineal preparation, while no evidence for a vasotocin-like peptide was obtained. The isolated neuropeptides contain a modified amino acid at position 2. This structural difference with authentic pituitary vasopressin and oxytocin may alter their biological and immunological properties, which have been interpreted as vasotocin-like, and thus underlies the controversy concerning the existence of vasotocin in the mammalian pineal gland.

Growth-inhibiting effect of crude pineal extracts on human melanoma cells in vitro is different from that of known synthetic pineal substances.

The effect was studied of a number of synthetic indoleamines, pteridines, beta-carbolines, of AVT and of crude extracts from rat and ovine pineal glands on human melanoma cells in vitro. The identified pineal substances as well as some of their analogues showed an inhibitory effect only at non-physiologically high concentrations. However, crude pineal extracts were more active than the synthetic pineal substances tested. They contain a compound which may have a tumor-inhibiting potency comparable to that of methotrexate but a different mechanism of action.

The effect of different photoperiods on the methylating capacity of the pineal gland of adult, male golden hamsters, with special reference to 5-methoxyindoles.

Testes weight, plasma FSH and LH concentration and pineal methylating capacity were compared in hamsters housed under either long (LD14:10) or short (LD8:16) photoperiods. Hamsters housed for 14 weeks under short photoperiod showed gonadal atrophy, which was complete after 6 weeks. Also plasma FSH and LH concentration showed a marked decline after transfer to short photoperiod. However, after 14 weeks the concentration of FSH and LH as well as testes weight increased again. Under both photoperiods day/night rhythms in plasma FSH and LH concentration were measured. Under both light regimes the concentrations did not show significant differences. Under long as well as short photoperiods in the pineal gland of animals no significant differences were found in the daily synthesis of various MI tested. Only the synthesis of ML was significantly higher in the pineal of hamsters housed under short photoperiod. The function of this higher synthesis of ML remains unknown. Although the maxima of the rhythm for the various MI found under different LD regimes did not differ in magnitude or duration, their location in respect to the onset of darkness was different. It is suggested that this specific location is of more physiological importance than the quantity or duration of synthesis, concentration or release of MI. At the moment the day/night rhythms were determined there were indications that recrudescence of the testes had already started. It is suggested that this recrudescence is responsible for the fact that no differences in the synthesis of MI were found comparing the influence of both photoperiods. After 14 weeks of exposure to short photoperiod, aML synthesis was, in contrast to the synthesis of the other MI, (not significantly) higher under LD8:16. Moreover, opposite results for aMT and aML synthesis during darkness were found. It is suggested that the ratio of synthesis of these compounds is of physiological significance.

Ultrastructural demonstration of secretion by exocytosis in rat pinealocytes with the use of the tannic acid method.

In the rat pineal gland the mechanism of release of secretory material was studied ultrastructurally after incubating tissues in Ringer solution containing tannic acid. The results indicate that pinealocytes release the contents of secretory vesicles into the extracellular space via exocytosis, a phenomenon that has not been visualized previously in this cell type. This finding may reflect release of polypeptides by the pineal gland.

Presence of immunoreactive neurophysins of a higher molecular weight in addition to the 10.000 form in the ovine pineal gland.

Using an aqueous extraction followed by ultrafiltration through Amicon Diaflo membranes, two ovine pineal fractions were obtained, which contain immunoreactive neurophysin. The presence of neurophysin was monitored by radioimmunoassay, employing an antiserum raised against pituitary bovine neurophysin and selected because it reacts with neurophysins of many other mammals. From 50 g of wet ovine pineal glands 552 micrograms of immunoreactive neurophysins were obtained. About 5% of these immunoreactive neurophysins are eluted from three different Sephadex columns with an elution volume corresponding to Mr above 10,000 between bovine serum albumin and pituitary neurophysin. The remaining 95% of ovine immunoreactive pineal neurophysin (Mr 10,000) shares immunological and physico-chemical properties with highly purified bovine pituitary neurophysin used as a reference. From the results of gel filtration and affinity chromatography on LVP-Sepharose it was concluded that ovine pineal gland may contain a neurophysin precursor molecule in addition to the neurophysin Mr 10,000.

The effect of ovine pineal compounds prepared under red or green light on the activity of male rat anterior pituitaries in vitro.

A high molecular weight fraction XM100R (MW A 100,000) was prepared by ultrafiltration from ovine pineals using two different extraction methods under red light conditions (lambda greater than 600 nm). This fraction stimulates the release of radioimmunologically active luteinizing hormone (LH) of anterior pituitaries in vitro. The ultrafiltration fraction PM30R (MW greater than 30,000 and less than 100,000) was found to be radioimmunologically active only when the "Bensinger" extraction procedure was applied. However, when comparable fractions were prepared under green light and incubated with half-pituitaries, all the incubation media of the ultrafiltrated fractions, XM100R, PM30R, PM10R (MW greater than 10,000 and less than 30,000) UM2R (MW greater than 1000 and less than 10,000), UM05R (MW greater than 500 and less than 1000) and UM05F (MW greater than 500), reacted with anti-LH. This may mean that under green light conditions the high molecular weight ovine pineal compounds in XM100R are disintegrated and/or split up into small molecules which can stimulate the release of LH, or crossreact with the anti-LH serum.

Rhythmic synthesis of various 5-methoxyindoles in the pineal gland of male adult golden hamsters, kept under the same artificial conditions throughout the year.

Until now the day/night and seasonal rhythmicity in the synthesis of 5-methoxyindoles (MI) is thought to be regulated by environmental factors, especially photoperiod and temperature. Endogenous factors are also implicated in the generation of N-acetyltransferase and hydroxyindole-O-methyltransferase activity rhythms. In the present experiments seasonal rhythmicity in the synthesis of MI in the pineal gland was investigated in hamsters kept under the same artificial conditions throughout the year. Though the environmental conditions were the same, day/night and seasonal rhythmicity in the production of MI in the pineal were observed indicating the existence of endogenous factors influencing the rhythmicities. In November, most of the MI showed the highest synthesis, MA and ML excepted, which were especially produced in July and September. The results obtained sustain the hypothesis that aMT is synthesized from MT rather than from aHT. Moreover, the rhythmicities in aMT synthesis are not identical to those found in aMT concentration as described in the literature. This indicates that synthesis and concentration of a compound are not comparable. At the end of the light period, when aMT injections have an antigonadotropic effect, a peak of aMT synthesis was always present. Although MI synthesis showed seasonal rhythmicity, no reproductive cycle occurred in the hamsters. At present, the concept that the pro- and/or antigonadal effects of the pineal are mediated by aMT seems to be the most acceptable. The present results, however, indicate that aMT and perhaps other MI, often regarded as factors influencing gonadal growth in golden hamsters, are not the only factors involved.