Showing papers in "Journal of Pineal Research in 1991"

Effects of melatonin on the cell cycle kinetics and “estrogen-rescue” of MCF-7 human breast cancer cells in culture

Abstract: Melatonin has been shown to have a direct inhibitory action on the proliferation of estrogen-responsive MCF-7 human breast cancer cells in culture. In the present study, we examined by flow cytometry whether this inhibitory effect might be exerted on the G1 phase of the cell cycle, thus causing a transition delay into the S phase. In order to further verify this hypothesis we tested the ability of estradiol to "rescue" MCF-7 cells from melatonin inhibition, and the potential of this indoleamine to block the ability of estradiol to rescue the cells from tamoxifen inhibition. Following five days of incubation, melatonin (10(-9)M) increased the fraction of cells in G1 of the cell cycle while simultaneously causing a 50% reduction in the proportion of cells in S phase. The antiproliferative effect of melatonin (10(-5)M) was prevented by the simultaneous treatment of the cells with estradiol (10(-8)M) in clonogenic soft agar culture, or reversed by the addition of estradiol to cells previously incubated with and inhibited by melatonin (10(-9)M) in monolayer culture. Additionally, melatonin blocked the estrogen-rescue of tamoxifen-inhibited cells in both types of culture systems. These results support the hypothesis that the antiproliferative effect of melatonin, like tamoxifen, is cell cycle specific by causing a G1-S transition delay. These results also indicate an important interaction of melatonin with estrogen-mediated mechanisms of MCF-7 cell proliferation.

Pineal gland "magnetosensitivity" to static magnetic fields is a consequence of induced electric currents (eddy currents).

Abstract: During the past decade, a number of reports indicated that the mammalian pineal gland is magnetosensitive in terms of spatial orientation. This indication is based on observations that artificial alterations of the direction of the earth's magnetic field (MF) markedly decreased the gland's capability to synthesize melatonin. These findings, however, seem paradoxical since animals as well as humans experience such alterations whenever they turn their heads. Therefore, the potential of the pineal for sensing magnetic fields was re-investigated. During the dark phase, rats were exposed to repeatedly inverted MFs, generated by two identical pairs of Helmholz coils; one pair connected to a power supply automatically, the other pair manually using an integrated potentiometer. Only the pineals of animals exposed to the automatically activated field responded with a reduced activity of the rate-limiting enzyme serotonin-N-acetyltransferase, lower melatonin levels and increases in serotonin and 5-hydroxyindole acetic acid. Hence, MF exposure itself did not affect the pineal. Rather, induced eddy currents in the animals, resulting from rapid On/Off transients of the artificially applied MF, are most likely the explanation.

Melatonin-induced enhancement of antibody-dependent cellular cytotoxicity.

Abstract: Antibody-dependent cellular cytotoxicity (ADCC) is a lytic mechanism in which a specific antibody acts cooperatively with leukocytic effector cells to induce target cell lysis. In this paper, the effect of exogenous melatonin on ADCC was examined. It was found that two evening intravenous injections of melatonin (1 mg/kg b.w.) was sufficient to enhance the capacity of splenocytes to mediate ADCC. This augmented activity returned to normal levels by day 6. Moreover, the opioid antagonist, naloxone, was unable to inhibit the ADCC enhancement, suggesting that melatonin did not operate through a naloxone-sensitive opiatergic mechanism. These results further support the modulatory action of melatonin on immune responses.

Urinary 6‐sulphatoxymelatonin, an index of pineal function in the rat

Abstract: The objective of this study was to apply the radioimmunoassay for 6-sulphatoxymelatonin (aMT6s) to rat urine, and use it to study the source of aMT6s. The radioimmunoassay was found to have acceptable within- and between-assay variation, excellent specificity, and good parallelism between the standard and unknown. Because urine is highly contaminated we assessed whether preliminary purification was required and established that it was unnecessary. Using this assay a 24-hr rhythm in 6-sulphatoxymelatonin output was seen in pools of urine harvested at 3-hr intervals from Wistar rats on LD 12:12. The nocturnal rise in aMT6s was abolished by constant light. In contrast pinealectomy lowered aMT6s output significantly throughout both dark and light. This study confirms previous studies indicating that the pineal is the major source of 6-sulphatoxymelatonin. It is concluded that urinary 6-sulphatoxymelatonin as measured by radioimmunoassay is a valid measure of pineal gland activity in the rat.

Melatonin-induced relaxation of rat aorta: interaction with adrenergic agonists.

Abstract: The influence of melatonin on alpha- and beta-adrenergic responses of vascular smooth muscle ex vivo were examined in these experiments. Melatonin caused a dose-dependent relaxation of precontracted (30 mM KCl) vascular smooth muscle. This response was not affected by the removal of vascular endothelium. Melatonin (10 nM) reduced the efficacy, but not potency, of the contractile responses to methoxamine and clonidine. Melatonin had no effect on the vascular beta-adrenergic response to isoproterenol. Pretreatment of vascular rings with lithium sulfate (0.1 mM) completely blocked the relaxation in response to melatonin, suggesting that the inositol phosphate pathway may be involved in this relaxation. Furthermore, pretreatment of vascular rings with phorbol 12-myristate-13-acetate, an activator of protein kinase C, antagonized the relaxation response to melatonin. Pharmacologic doses of melatonin (0.1 mM) slightly impaired the vascular smooth muscle response to calcium. These data indicate that melatonin per se is capable of relaxing vascular smooth muscle and that low doses of melatonin impair alpha-1 and alpha-2 adrenergic responses without changes in the beta adrenergic response of vascular smooth muscle.

Circadian patterns of melatonin, corticosterone, and progesterone in male rats subjected to chronic stress: effect of constant illumination.

Abstract: Plasma and pineal melatonin and plasma corticosterone and progesterone concentrations have been shown to be altered by several types of stressors. This study was designed to define the circadian patterns of the hormones mentioned above in rats subjected to chronic stress and to investigate the influence of constant illumination. The results revealed that melatonin and corticosterone circadian patterns deteriorated and their plasma concentrations were significantly elevated. The constant illumination (2,500 lux) during the dark period (from 2000 to 0600) was not able to suppress melatonin production in stressed animals, while the plasma content of corticosterone was decreased at the end of experimental period compared to control rats. Plasma levels of progesterone were increased in stressed animals as well. Constant illumination, however, provoked also an increase of progesterone secretion in controls. Statistical comparisons between hormonal secretory patterns showed that melatonin and corticosterone correlated negatively in controls (r = -0.58, P less than 0.05) during the nighttime. However, in stressed animals correlation was observed only between melatonin and progesterone secretion during the light and dark period (r = -0.43, P less than 0.05). Surprisingly, the correlation during the nighttime in rats subjected to constant illumination was negative (r = -0.60, P less than 0.02) compared to positive correlation (r = 0.60, P less than 0.02) in rats kept under normal lighting regimen. These results suggest that melatonin release is affected by stress and, possibly, under these circumstances, interacts with adrenal steroid secretion.

Melatonin, a suppressor of spontaneous murine mammary tumors.

Abstract: The effect of prolonged, oral melatonin treatment on spontaneous mammary tumor development in female C3H/Jax mice was studied. Melatonin was administered at a dose of 25 micrograms/mouse/day from 21 to 44 days of age and 50 micrograms/mouse/day from day 45 onwards. By the age of 12 months, 62.5% of control animals developed tumors as opposed to 23.1% in the melatonin treated group (P less than 0.02). In both control and treated mice, the thoracic pairs of mammary glands were obviously more susceptible to spontaneous mammary tumor development, as at least 50% of the total tumors developed in this region. Reduction in submaxillary and pituitary gland weights of treated animals was observed at necropsy (P less than 0.001). Decreased serum 17-beta-estradiol (E2) levels in melatonin treated mice (P less than 0.05) and a marked reduction in [3H]thymidine incorporation into DNA of melatonin-treated mammary glands (P less than 0.02) positively correlated with the sparse mammary gland development seen in these mice. These observations indicate that at the given dose level, melatonin modulates the degree of development of the mammary epithelium, subsequently reducing spontaneous mammary tumor incidence in these high risk mice.

Immunohistological localization of melatonin in the pineal gland and retina of the rat.

Abstract: An incubation procedure for the demonstration of melatonin is described using freeze-dried, unfixed material embedded in vacuum directly in glycolmethacrylate. The results indicate the binding of melatonin in the nucleus of pineal parenchymal cells and in the outer nuclear layer of the retina of rats; binding is greater at night than during the day.

Influence of pineal indoleamines on the mitotic activity of gastric and colonic mucosa epithelial cells in the rat : interaction with omeprazole

Abstract: We have investigated the effects of melatonin (Mel) and N-acetylserotonin (NAc-5HT) on the mitotic activity of gastric and colonic mucosa in adult male rats under basal conditions and after an administration of omeprazole (OM) (H+,K(+)-ATPase inhibitor). The metaphase-arrest technique was applied in the study. Additionally, serum gastrin levels were measured by RIA method in the OM-treated group and in respective polyethyleneglycol (PEG)-administered controls. We have found that: 1) OM-treatment increased serum gastrin levels in rats; 2) OM enhanced the mitotic activity of the colonic mucosa cells, although, unexpectedly, it did not exert such an effect on the gastric mucosa cells; 3) Mel suppressed the OM-induced increase of the colonic epithelium cell proliferation, while NAc-5HT failed to reveal that action: 4) NAc-5HT decreased the proliferation of gastric mucosa epithelial cells. The value of the mean mitotic activity rate (MMAR) of gastric mucosa after Mel-treatment also decreased, but that change was not statistically significant. The obtained data are in compliance with previous results from our laboratory concerning the inhibitory effect of pineal indoleamines on the jejunal epithelium mitotic activity. The stimulatory effect of OM on the proliferation of colonic epithelium is probably mediated by OM-induced hypergastrinaemia. The possibility of Mel interaction with intestinal gastrin receptors (a structural similarity occurs between Mel and benzotript, a specific gastrin receptor antagonist), as well as of the opposite effects of Mel and gastrin on intracellular cAMP content in the gut, are considered in the discussion of results.

Insulin release from isolated mouse islets in vitro: No effect of physiological levels of melatonin or arginine vasotocin

Abstract: Some data in the literature suggest that heightened activity of the pineal gland may be diabetogenic. The onset of insulin-dependent diabetes mellitus is highest during the winter months and at puberty when melatonin levels are also greatest. To study the direct effects of pineal hormones on insulin release, hand-dissected ob/ob-mouse islets of Langerhans were incubated in vitro with melatonin (1 nmol/l to 100 mumol/l) or arginine vasotocin (1 pmol/l to 10 mumol/l) and D-glucose (3 or 20 mmol/l for 1 hr. Melatonin did not affect basal or glucose-stimulated insulin release. Arginine vasotocin (AVT) did not affect basal insulin release, but at presumably pharmacological levels (1 and 10 mumol/l) the peptide significantly increased glucose-stimulated insulin release. We conclude that melatonin and AVT at physiological concentrations have no direct effect on islet insulin release, and that any diabetogenic effect of the pineal gland must occur via suppression of insulin action or via production of a metabolite or hormone that suppresses insulin release.

Effects of different doses and durations of melatonin infusions on plasma melatonin concentrations in pinealectomized Syrian hamsters: consequences at the level of sexual activity.

Abstract: The effect of different doses and durations of melatonin infusions on plasma melatonin concentrations has been studied in pinealectomized Syrian hamsters maintained under short photoperiod at either 7 degrees C or 18 degrees C. The effects of the infusions on plasma melatonin concentrations and on gonadal activity were compared. The results show that the minimal effective quantity of infused melatonin that induced gonadal atrophy was 40 ng/h at 7 degrees C and 20 ng/h at 18 degrees C. An infusion of 8 hr duration per day is necessary to inhibit sexual activity, while an infusion of 6 hr duration was ineffective. This finding suggests that the critical duration of melatonin infusion is between 6 and 8 hr. Despite the various doses of melatonin infused, plasma melatonin concentrations measured in the middle of the infusion period did not differ significantly from concentrations measured in intact animals. This finding suggests that the metabolism of infused melatonin increases as the dose of melatonin increases. Moreover, the different physiological effects observed after the various melatonin infusions cannot be explained by variations in plasma melatonin concentrations.

Regulation of melatonin production by catecholamines and adenosine in a photoreceptive pineal organ. An in vitro study in the pike and the trout.

Abstract: The pineal organ of fish contains photoreceptor cells with structural and functional analogies to retinal photoreceptors. In these cells, the light/dark (LD) cycle influences the production of melatonin by controlling the activity of one of its synthetizing enzymes, serotonin N-acetyltransferase (NAT). The daily rhythm in NAT activity is generated endogenously in the pike but not in the trout pineal. We report here that in addition to the LD information, chemical factors are also involved in the control of melatonin production. Adenosine and two of its analogs stimulated or inhibited NAT activity and melatonin release in cultured pike and trout pineals, depending on the experimental conditions. It is believed that the nucleoside, produced locally, exerts a modulatory role on the neurohormonal output via still enigmatic mechanisms, involving a transmembranous carrier. Nocturnal melatonin production in cultured pike pineals was inhibited by alpha-adrenergic agonists and stimulated by a beta-adrenergic agonist. No effect could be induced in trout pineals cultured under similar conditions. Because melatonin production by pineal photoreceptors is apparently regulated by both light and chemical inputs, we propose they might be multieffector cells.

Seasonal effects of administration of melatonin and 5-methoxytryptophol on ovarian activity in the catfish Heteropneustes fossilis (Bloch).

Abstract: k >y KP, Agha AK. Seasonal effects of administration of melatonin and 5-methoxytryptophol on ovarian activity in the catfish Heteropneustes,ti,ssilis (Bloch). J Pineal Res 1991:10:65-70. Abstract: In Heteropneustes fossilis, effects of intraperitoneal administration (25 fig/fish, alternate days for a month 1 hr before the onset of dark phase) of melatonin (MEL) and 5-methoxytryptophol (5-MTL) on ovarian activity were investigated during different seasons of the annual reproductive cycle under natural photothermal conditions. The gonadosomatic index (GSI) had decreased significantly by MEL during preparatory and prespawning seasons, and by 5-MTL in all but the spawning season. Both MEL and 5-MTL administrations arrested vitellogenesis during the gonadal recrudescence phase. As a result, stage I previtellogenic oocytes showed a significant increase in the MEL group in the preparatory and prespawning seasons, and in the 5-MTL group only in the prespawning season. Almost the same trend was noticed in the percentage distribution of stage 2 and 3 oocytes. The percentage distribution of stage 4 postvitellogenic oocytes decreased significantly during the preparatory and prespawning seasons, but not in the spawning season. The indole treatment resulted in a highly significant increase in the percentage distribution of atretic oocytes during prespawning, spawning, and post-spawning phases. Total protein content of the ovary was significantly lower in all seasons after MEL treatment, and only in the preparatory and prespawning seasons following 5-MTL treatment. The ovarian glycogen level was significantly lower in the preparatory and prespawning seasons (MEL group), and in the 5-MTL group only in the prespawning phase. Ascorbic acid level was inhibited by the indole treatment in all seasons except in the 5-MTL group during the postspawning season. The results show that both MEL and 5-MTL are effective in inhibiting ovarian vitellogenesis and inducing atresia in the catfish.

Plasma melatonin in the adelie penguin (pygoscelis adeliae) under continuous daylight in antarctica

Abstract: Circadian rhythms of melatonin secretion in birds are influenced by daylength and light intensity. Daily patterns of melatonin secretion were examined in Adelie penguins (Pygoscelis adeliae) under natural continuous daylight at Cape Bird, Antarctica (77 degrees S). Although daylight is continuous during the Antarctic summer there was a marked daily cycle of light intensity. However, there was no relationship between mean plasma melatonin levels and time of day in groups of 2-10 penguins sampled at 2-3 h intervals in November, December, or January. Mean melatonin levels over 24 h in groups of birds from which single samples were collected, or in groups of birds sampled repeatedly through cannulae, were low (12.4 +/- 1.2 pg/ml-28.8 +/- 4.4 pg/ml for 4 sampling periods; n = 22-163). Levels in individual birds were, however, quite variable and ranged from 5.0-68.1 pg/ml. Some birds had periods of increased melatonin levels that tended to occur during the time of day when light intensity was least. One bird had a clear low amplitude melatonin rhythm with a peak during the time of least light intensity. These results, the first for any bird under a natural photoperiod, indicate that melatonin secretion is inhibited by natural continuous daylight, but that it is not abolished.

Inhibition of human platelet aggregation and thromboxane B2 production by melatonin. Correlation with plasma melatonin levels

Abstract: Plasma melatonin concentrations and the effect of melatonin on arachidonic acid (AA)-induced aggregation and thromboxane B2 (TxB2) production by platelet-rich plasma (PRP) were examined in five normal male volunteers, sampled at 2 hr intervals from 21:30 to 09:30 hr. Peak plasma melatonin concentration was found at 03:30 hr. Inhibition by 10(-6) M melatonin of AA-induced PRP aggregation was observed only in samples taken at 01:30 hr. Assessment of the inhibitory effect of 10(-9)-10(-6) M melatonin on AA-induced TxB2 production indicated that melatonin activity was greater at 01:30 h as compared to late night. Assessed as a global effect, the inhibitory activity of melatonin on PRP TxB2 showed a maximum at 01:30 hr and minimal effects at 03:30 hr, at the time when plasma concentrations of melatonin were highest. These results indicate the existence of a nocturnal variation in sensitivity of human platelets to melatonin, with a peak that precedes the maximum in circulating melatonin levels.

Pineal-gonadal relationship in the teleost Channa punctatus (Bloch): evidence for possible involvement of hypothalamic serotonergic system.

Abstract: In Channa punctatus, pinealectomy results in an accelerated growth (stimulatory effect) of the ovary in preparatory phase (March), but had no significant effect in prespawning (May-June) or postspawning (September) phases. Administration of melatonin (25 micrograms/fish or 0.4 mg/kg BW at 2-day intervals for 30 days) inhibits the stimulatory effect of a long photoperiod and high temperature (16L:8D; 22 +/- 1 degree C) on the ovary in the early preparatory phase (February). In the late preparatory phase (April), the administration of both melatonin (0.5 mg/kg BW at 2-day intervals) and parachlorophenylalanine (pCPA, a serotonin synthesis blocker; 100 mg/kg BW at 3-day intervals) for 30 days inhibits ovarian activity in comparison to that of the saline-treated control group. In the dose-response study, greater than or equal to 0.5 mg/kg BW of melatonin induced a significant increase in hypothalamic 5-HT content and greater than or equal to 10 mg/kg BW of pCPA decreased it significantly. In the time-course study, melatonin (2.0 mg/kg BW) elevates the 5-HT content significantly after an hr of the injection and maintained it up to 48 hrs. The administration of pCPA (200 mg/kg BW) had significantly inhibited the 5-HT content which was sustained for 72 hr. In another study, a single injection of melatonin (0.5 mg/kg BW) increased the 5-HT content significantly. A single injection of pCPA (100 mg/kg BW) decreased significantly both the content and activity of 5-HT. It is inferred that hypothalamic 5-HT may play a central role in photosexual mechanisms and mediate long photoperiodic effects on neuroendocrine-reproductive axis.

Melatonin effect on arachidonic acid metabolism to cyclooxygenase derivatives in human platelets.

Abstract: The effect of melatonin on thrombin-induced [3H]-arachidonic acid (AA) metabolism to cyclooxygenase derivatives was determined in platelets obtained from normal volunteers at 0830 and 2030 h. Percent conversion of radioactive AA was generally greater at 2030 h than at 0830 h for every cyclooxygenase derivative analyzed. Micromolar or greater concentrations of melatonin decreased significantly the conversion of [3H]-AA to prostaglandin (PG) F2 and thromboxane (Tx) B2, and inhibited slightly the conversion to PGE2 and PGD2. After preincubation of platelets with 1 mM imidazole, the melatonin inhibitory effect was significant for PGF2 only. Melatonin (10(-6) M) showed a significant inhibitory influence on platelet ATP release induced by phorbol-12 myristate-13 acetate (PMA) at 2030 h, an effect inhibited by 1 mM aspirin. These results indicate that at pharmacological concentrations melatonin inhibits human platelet cyclooxygenase.

Effect of a 15 minute light pulse on nocturnal serum melatonin levels in human volunteers.

Abstract: Petterborg LJ, Kjellman BF, ThalQ BE, Wetterberg L. Effect of a 15-minute light pulse on nocturnal serum melatonin levels in human volunteers. J Pineal Res 1991:10:9-13. Abstract: Monitoring the daily melatonin rhythm during the course of phototherapy for affective disorders may be beneficial in assessing the efficacy of such treatments. It is therefore of interest to study the effects of the timing, duration, and intensity of bright light pulses on melatonin levels in normal subjects. To examine the effects of a single exposure to a brief burst of bright light on serum melatonin, groups of healthy human volunteers of both sexes were treated with a 15 minute pulse of bright light (350 cd/m2) early in the evening during the winter months. Serial blood samples were collected from each person and the effect of the light pulse on serum melatonin and cortisol levels determined. Melatonin levels were significantly but only transiently suppressed by the light pulse, while cortisol levels were not affected. These results demonstrate that short duration bright light treatments can influence the melatonin rhythm generating system in humans.

Pineal influence on the diurnal rhythm of nonspecific immunity indices in chickens.

Abstract: The effect of pinealectpmy and melatonin injections on the diurnal rhythms of serum lysozyme and blood granulocytes was examined in White Leghorn cockerels kept from time of hatching for 5 weeks in L:D 12:12 conditions and immunized twice with sheep red blood cells (SRBC). Pinealectomy or sham-operation was made during first week of life. Pinealectomized chickens were injected daily with a melatonin dosage increased over 4 consecutive weeks (the dosage was 10, 13, 16, and 20 ng per bird daily during the 4 weeks, respectively; MEL I) at the beginning of darkness. The same treatment was performed on chickens with an intact pineal gland using additional melatonin doses increased 10 times (MEL II) and 500 times (MEL III). Intact chickens were also injected with MEL II and MEL III 4 hr before the end of light. Control birds received equivalent injections of vehicle. Five-week-old chickens were sacrificed during a 24-hr period every 4 hr. The existence of diurnal rhythm was evaluated by cosinor analysis. Pinealectomy shifted the acrophase of the diurnal rhythm of granulocytes and abolished that of serum lysozyme. Both rhythms were restored in pinealectomized chickens by MEL I but not by vehicle injections. The same melatonin dose was unable to change the granulocyte rhythm but delayed the acrophase of that of serum lysozyme in chickens with an intact pineal gland. Two higher melatonin doses influenced the diurnal rhythm of granulocytes as a function of dose and time of administration. The rhythm of serum lysozyme was dependent only on the time of injection. The pineal gland seems to control, via its hormone melatonin, the diurnal rhythm of nonspecific immunity in chickens.

5-Methoxypsoralen enhances plasma melatonin concentrations in the male rat: non-noradrenergic-mediated stimulation and lack of effect in pinealectomized animals.

Abstract: Previous studies have shown that 8-methoxypsoralen (8-MOP) is able to enhance melatonin secretion from rat pineal glands. The effect of another psoralen, 5-methoxypsoralen (5-MOP) on plasma melatonin concentrations alone or in combination with isoproterenol (Iso, 10 mg/kg BW, i.p.) or propranolol (Prop, 20 mg/kg BW, s.c.) was studied in the rat. An increase in plasma melatonin concentrations was observed 1 h after psoralen administration and lasted 8 h. The stimulatory effect of 5-MOP on circulating melatonin concentrations was dose dependent above 5-mg/kg, BW. 5-MOP also potentiated the Iso-induced rise in plasma melatonin levels. Furthermore, Prop did not block the effect of 5-MOP, indicating that the psoralen acts independently of the noradrenergic system controlling pineal melatonin synthesis. No increase in plasma melatonin levels was observed in pinealectomized (Px) animals after injection of 5-MOP (20 mg/kg BW) or 8-MOP (20 mg/kg BW). This demonstrates that the rise in plasma melatonin concentrations observed after treatment requires the presence of the pineal gland, and that neither 5-MOP nor 8-MOP stimulates melatonin release from other structures such as the retina or Harderian gland.

Localization and quantification of high-affinity melatonin binding sites in Rana pipiens retina.

Abstract: Melatonin binding was localized to the inner plexiform layer (IPL) of the frog retina by in vitro autoradiography, using 2-125I-melatonin as the radioligand. Radioreceptor binding assays of frog retinal homogehate demonstrated saturable melatonin binding. Scatchard analysis revealed a single population of binding sites with an apparent dissociation constant (Kd) of 125 pM, with a Bmax of 0.138 fmoles/mg of protein. These results suggest that high-affinity melatonin binding sites are present in the IPL of the frog retina, which may reflect the presence of melatonin receptors in this synaptic layer.

GABA‐immunoreactive intrinsic and ‐immunonegative secondary neurons in the cat pineal organ

Abstract: Vigh-Teichmann I, Petter H, Vigh B. GABA-immunoreactive intrinsic and 4mmunonegative secondary neurons in the cat pineal organ. J Pineal Res 199 1 : 10: 18-29. Abstract: The pineal organ of the cat was studied by postembedding gamma-aminobutyric acid (GABA) immunocytochemistry. Two polyclonal rabbit GABA antisera were used with light microscopic peroxidase and electron microscopic immunogold techniques. A considerable number of intrinsic neurons are scattered in the proximal portion of the pineal organ. Some of the nerve cells were GABA-immunoreactive; other neurons as well as pinealocytes and glial/ependymal cells were immunonegative. A few GABA-immunoreactive neurons behaved like CSF-contacting neurons by penetrating the ependymal lining of the pineal recess. GABA-immunoreactive neurons were more frequently found in the subependymal region. Small bundles of thin immunoreactive unmyelinated and thick immunoreactive myelinated nerve fibers occurred in the proximal pineal, especially near the habenular commissure. There were synapses of various types between GABA-immunoreactive and -immunonegative fibers. Myelinated immunoreactive axons seemed to loose their sheaths after entering the organ. Axon-like processes of pinealocytes terminated on dendrites of immunonegative neurons present near the posterior and habenular commissures. The axons of these neurons were found to join the commissural fibers and may represent a pinealofugal pathway conducting information originating from pinealocytes. The pinealocytic axons forming ribbon-containing synapses on dendrites of secondary neurons speak in favor of the sensory-cell nature of the pinealocytes. The pinealopetal myelinated GABA-immunoreactive axons and the intrinsic “GABA-ergic” neurons are proposed to inhibit the action of intrapineal neurons on which the pinealocytic axons terminate.

The effect of pinealectomy and autonomic denervation on crypt cell proliferation in the rat small intestine.

Abstract: Previously it has been found that pinealectomy in rats was associated with increased small bowel crypt cell proliferation. The hypothalamus is thought to play a role in the neural control of crypt cell proliferation and the pineal gland is known to be functionally connected with this component of the autonomic nervous system. Consequently, the effect on crypt cell mitotic rate of pinealectomy with autonomic denervation of the rat small bowel (local sympathectomy and truncal vagotomy) was observed to determine the possible importance of the autonomic nervous system in the mediation of the effects of pinealectomy on the crypts. It was found that interruption of either the vagal or sympathetic nerve supply to the small intestine both significantly decreased the usual hyperproliferative effect on the crypt cells observed after pinealectomy. It is suggested that the overall effect of the pineal gland is to suppress the crypt cell proliferation rate and that this effect is mediated, at least to some extent, by (1) the direct connections between the pineal gland and the hypothalamus, (2) the connections between the hypothalamus and the vagal and sympathetic innervation of the gut, and (3) the influence of the autonomic innervation on the activity of the enteric plexuses in relationship to the crypts. The pineal may have a role in modulating the local control mechanism of crypt cell proliferation. Its exact role in the control of crypt cell proliferation has not yet been defined.

Chronic administration of melatonin induces changes in porphyrins and in the histology of male and female hamster harderian gland: interrelation with the gonadal status.

Abstract: In this paper, we have investigated the influence of melatonin on the histology and porphyrin content of the Syrian hamster Harderian glands. Daily afternoon injections of 25 micrograms of melatonin to female hamsters for 12 weeks resulted in the discontinuity of estrous cyclicity, a marked decrease in the Harderian gland intraluminal area occupied by porphyrins, and in a significant rise in the number of Type II cells. A similar decrease in porphyrins was observed after 8 weeks of ovariectomy. However, if the melatonin injections were given for only 8 weeks (without inducing gonadal atrophy), no changes were observed in the area occupied by intraluminal porphyrins, suggesting that the effects of melatonin in female Syrian hamsters might be associated with the subsequent gonadal atrophy. Castration of male hamsters induced a significant increase in porphyrins and a clear drop in the number of Type II cells. These changes were totally prevented when melatonin was administered daily from the day of castration. Our results suggest that melatonin, at least in male Syrian hamsters, plays a role in Harderian metabolism, acting directly on the Harderian secretory cells or indirectly through pituitary hormones.

Differential effects of pinealectomy on amygdala and hippocampus serotonin metabolism.

Abstract: Miguez J, Martin F, Aldegunde M. Differential effects of pinealectomv on amygdala and hippocampus serotonin metabolism. J Pineal Res 1991 : 10: 1 W103. Abstract: The purpose of the present study was to determine the effects of long-term pinealectomy on serotonin metabolism in the amygdala and the hippocampus of male rats. Pinealectomy did not significantly alter either tryptophan or serotonin concentrations in the amygdala or the hippocampus. However, statistically significant decreases in 5-hyroxyindole-3-acetic acid levels and tryptophan hydroxylase activity were found in the amygdala. Monoamine oxidase activity was unchanged in both regions. These results support the involvement of the amygdaloid serotoninergic system in mediating the functions of the pineal gland.

Long‐term pinealectomy alters hypothalamic serotonin metabolism in the rat

Abstract: In the present study, the effects of long-term pinealectomy on tryptophan, 5-hydroxytryptamine (5-HT or serotonin), 5-hydroxy-3-indoleacetic acid (5-HIAA), and tryptophan hydroxylase and monoamine oxidase activities were studied in preoptic area-anterior hypothalamus (POA-AH) and in the medial and posterior hypothalamus of the rat. After pinealectomy, 5-HT levels decreased significantly in medial hypothalamus but increased in the POA-AH. The levels of 5-HIAA decreased significantly in the POA-AH and medial hypothalamus. Tryptophan levels remained unchanged while tryptophan hydroxylase activity diminished significantly in POA-AH and medial hypothalamus. Monoamine oxidase activity remained unchanged in the hypothalamic regions. These results suggest that pinealectomy induces differential inhibitory actions on the serotoninergic terminal regions, mainly in anterior and medial hypothalamic areas.

Identification of serotonin 5HT2 receptors in bovine pineal gland.

Abstract: The concentration of serotonin in the pineal gland is extremely high, which prompted speculation that in addition to serving as a precursor of melatonin, serotonin may have an independent function of its own. By using [3H]-spiperone as a ligand, and ketanserine as a selective serotonin 5HT2 receptor antagonist, we have identified 5HT2 receptor in the bovine pineal gland, revealing a single population of binding sites with a dissociation equilibrium constant (Kd) value of 1.26 +/- 0.41 nM and a receptor density (Bmax) value of 193 +/- 38.85 fmol/mg protein. In displacement experiments, the concentrations of the drugs required to inhibit 50% of the specific binding of [3H]-spiperone in descending order of potency were methysergide greater than ritanserin greater than pirenperone greater than pipamperone greater than ketanserin greater than cyproheptadine greater than M-trifluoromethylphenyl-piperazine greater than prazosin greater than 5-methoxy-N-N-dimethyltryptamine hydrogen oxalate greater than 1-(3-chlorophenol) piperazine greater than serotonin. In the rat pineal gland, [3H]-spiperone revealed a low affinity serotonin binding site with a Kd value of 25.77 +/- 10.7 nM and a Bmax value of 1244 +/- 472 fmol/mg protein. The results of these studies are interpreted to indicate that the bovine pineal gland possess serotonin 5HT2 receptor. However, the rat pineal gland possess a serotoninergic binding site of unknown nature.

Ultrastructure and opsin immunocytochemistry of the pineal complex of the larval Arctic charr Salvelinus alpinus: A comparison with the retina

Abstract: The fine structure and opsin immunocytochemistry of the pineal and parapineal organs of the salmonid fish Salvelinus alpinus, the landlocked Arctic charr, were studied and compared with the retina in various developmental stages, from prehatching to two-month-old. For opsin immunocytochemistry two polyclonal antibovine rhodopsin and the monoclonal antichicken opsin antibodies OS-2 (detecting blue and green pigments) and OS-1 (detecting green and red pigments) were used. Histologically, the pineal organ consists of nervous tissue like that of the retina. It is composed of photoreceptor pinealocytes, which formed axon terminals containing synaptic ribbons, on the dendrites and perikarya of secondary pineal neurons. Already in prehatching embryos, both the pineal and retinal photoreceptors display well-developed outer segments and form synaptic terminals. The distal part of the pineal organ differentiates earlier than its proximal stalk. The differentiation of the retina starts centrally, but the caudal and dorsal retinae are differentiated earlier than the rostral and ventral ones. At the end of the larval period, the lateral retina is still undifferentiated. In all stages studied, (rhod)opsin immunoreactivity was found in the outer segments of the pineal organ and rod-type retinal photoreceptors, a finding speaking in favour of the presence of the opsin of a rhodopsin/porphyropsin. Cone-type retinal photoreceptors identified morphologically in the pre- and posthatching stages were opsinimmunonegative with the four primary antisera used. This result suggests that in the charr the opsins of cone visual pigments differ in their chemical nature from those of rhodopsin/porphyropsin. The parapineal organ was opsin immunonegative. Using the monoclonal antibody OS-2 opsin immunoreactivity was also detected in inner segments, perikarya, and pedicles of rod-type photoreceptors of both retina and pineal organ of embryos and 1- to 4-day-old larvae. This may indicate a high level of opsin gene expression during photoreceptor growth around hatching. The well-developed pineal organ and its opsin content are discussed in connection with the photonegative behaviour of the larval charr.

Effect of dose and time of melatonin injections on the diurnal rhythm of immunity in chicken.

Abstract: Skwado-Solita K, Thaela M-J, GXuchowska B, Stepien D, Jagura M. Effect of dose and time of melatonin injections on the diurnal rhythm of immunity in chicken. J Pineal Res 1991: 10:3&35. Abstract: The effect of daily melatonin injections on the diurnal rhythm of immune parameters was examined in White Leghorn cockerels, kept from hatching in L:D 12:12 conditions. Subcutaneous injections of melatonin were made at the beginning of darkness or 4 h earlier for four weeks starting from one week of life. The melatonin dosage in one group was raised (10, 13, 16, and 20 ng per bird daily, respectively) during four consecutive weeks. The two other doses were 10 and 500 times higher and were increased every week as well. Control birds received equivalent injections of vehicle. Three-week-old chickens were immunized ip with sheep red blood cells and reimmunized one week later. Five-week-old birds were sacrificed during a 24 h period every 4 h. The existence of the diurnal rhythm was evaluated by cosinor analysis. The diurnal rhythm of total white blood cells and serum agglutinins was more dependent on the time of melatonin injections than on the hormone used. The effect of melatonin injections on the level of immune parameters examined was also dependent on the time of sample collection. Results obtained indicate the participation of pineal gland in the regulation of the diurnal rhythm of the examined indices of avian immune system function that exhibit diurnal changes in sensitivity to exogenous melatonin.

Circadian and seasonal rhythms of melatonin production in mules (equus asinus equus caballus)

Abstract: The present study describes the patterns of melatonin production in the mule (Equus asinus x Equus caballus). Blood was sampled hourly for 24 h from eight mule mares in spring and fall. The data obtained show the presence of a circadian rhythm of production of melatonin, with highest values during the dark phase both in spring and fall. In fall the nightly rise of melatonin production begins earlier in the dark phase and reaches higher quantitative levels than in spring. The morning decline of melatonin production is similar in the two seasons. Maximal levels of nightly melatonin production in the mule are about 10 x higher than those described in the horse. The results reported here indicate the persistence of brain structures able to receive and transduce environmental signals in the mule, a genetically sterile mammalian hybrid.