Pineal Indoles Research Articles

Electrical responses of pineal cells to pineal indoles and putative transmitters in intact and blinded pigeons.

The effects of micro-electrophoretically applied pineal indoles, melatonin (MEL), 5-methoxytryptophol (MTL) and 5-hydroxytryptophol (HTL) and of the neurotransmitters, noradrenaline (NOR), acetylcholine (ACH) and gamma-aminobutyric acid (GABA) on the electrical activity of pineal cells were studied in anaesthetized pigeons. Recordings were made from both sighted and blinded birds during both the day- and night-time. Both excitatory and inhibitory responses to the application of all substances tested were observed, while in all cases unresponsive cells were also found. The pattern of responses to MEL, MTL and to NOR varied significantly depending on whether the cells in the sighted pigeons were tested during the day or at night. This day/night rhythm of responses to MEL and to MTL also occurred in the blinded pigeons, although this was not the case for the effects of NOR. Units which were orthodromically excited by electrical stimulation of the habenular complex were also excited by application of GABA. The activity of these units was also likely to be enhanced by application of ACH. The results suggest that the pigeon pineal organ exhibits intrinsic circadian rhythmicity which does not require entrainment signals from the retina. They also indicate that the activity of pineal cells could be influenced by neurotransmitter input provided from within the gland or via its innervation.

Day/night differences in pineal indoles in the adult pigeon ( Columba livia)

1. Day/night differences in concentrations of 5-hydroxy and 5-methoxy indole metabolites in the pineal gland of the pigeon are described. 2. A simultaneous determination of 5-hydroxytryptamine (serotonin), 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, N-acetyl-5-hydroxytryptamine ( N-acetyl serotonin), 5-methoxyindoleacetic acid, 5-methoxytryptophol, tryptophan, indoleacetic acid and melatonin was accomplished using a recently developed procedure employing high-performance liquid chromatography with electrochemical detection. 3. As in mammalian species, an inverse relationship was observed between N-acetylated indoles and serotonin and its acid metabolites. 4. Melatonin and N-acetyl serotonin were increased approximately three-fold at night to concentrations of 0.730 and 1.79 ng/pineal respectively. 5. Daytime serotonin values were 44.9 ± 13.0 ng/pineal and decreased to 12.3 ± 6.5 ng/pineal during the dark phase.

Prostaglandins and the regulation of motility in non-pregnant isolated uterus

Introduction Long before the prostaglandins were identified and recognized as a novel family of biologically active compounds, Kurzrok and Lieb reported the fwst observations of the influences of semen on human uterine contractionsL With hindsight, it is rrtost likely that the effects detected were evoked by prostaglandins present in the seminal fluid. Various pioneer studies showed that exogenous prostaglandins (particularly of the E and F series) potently affected con. tractions of the uterus from several speciesE Some years later it was shown that prostaglandins were released by distention of the uterus and several lines of experimentai evidence strongly suggested that during pregnancy endogenous uterine prostaglandins were able to induce and modulate myometriai activity. In 1973 Vane and Williams' reported that uteri isolated from pregnant rats exhibited intermittent spontaneous contractions and released a prostaglandin-like substance (mainly similar to PGF,.) into the bathing fluid. Both the output of the material and the contractile activity were abolished by indometacin, meciofenamate or acetylsalicylic acid. it was also found that low concentrations of PGF,2 or PGFa,, restored contractions of uteri rendered quiescent by inhibitors of prostaglandin synthetase a. Although until this time it was not gener. ally accepted that prostaglandins generated by the non.pregnant uterus could be effective modulators of its motility, some precedents favoured the contention s-8. The pre. vailing notion was that intramural prostaglandin synthesis by the pregnant rat uterus (and presumably by the non.pregnant uterus as well) was paralleled by tissue spontaneous contractile activity. It must be noticed that, as indicated by Piper and Vane, prostaglandin release can be equated with prostaglandin synthesis, in any event, one question was still unanswered, i.e. whether the generation and release of prostaglandins from the uterus should be considered as a consequence, rather than as a cause of contractions. The answer was not long in arriving. Prostaglandin output from the uterus occmred even when tissue motility was abolished by papaverine, a potent smooth muscle relaxant, precluding the possibility of prostaglandin production and release as a consequence of uterine contractions. Further links be!ween tissue prostaglan. din generation and contractions of the non.pregnant uterus were demonstrated by more recent experiments in which the effects of melatonin, considered as one of the major secretory products of the pineal gland, were tested on prostaglandin output and motility of isolated rat uterus e. The rationale for these studies, based on the structural similarities between melatonin and the prostaglandin inhibitor, indo. metacin, prompted us to examine the possibility that melatonin and indometacin could share biological properties, it was then observed that the pineal indole (10 -s M, or higher) inhibited prostaglandin E and F release and the spontaneous contractions of the rat uterus isolated from ovariectomized rats, as well as its reactivity to added oxyto. cin. lvlelatonin effects on uterine motility coulc!, there,fore be associated with the inhib. ition of prostnglandin synthesis, and the lack of action of oxytocin may be interpreted as resulting from the impairment of its influence on uteri containing low pros. taFlandin levels a. Moreover, the inhibition of contractions was similar to that observed by other investigators and ourselves after incubation with indometacin'.'. On the other hand, the negative inotropic effect was overcome by deSvering POF,., a finding which suggested that the contractile apparatus of the tissue remained undamaged following melatonin incubation.

Natural and synthetic analogues of melatonin and related compounds. II. Effects on plasma thyroid hormones and cholesterol levels in male Syrian hamsters.

Synthetic or natural analogues of the pineal indole, melatonin, were injected separately every evening (1700 hours) for 7 (Exp. 1) or 10 (Exp. 2) weeks into adult male Syrian hamsters maintained in 14 hours of light and 10 hours of darkness each day. Plasma thyroxine (T4) levels were significantly depressed by 25 micrograms/day of melatonin (aMT) in both experiments. Injecting 25 micrograms/day either of acetyl methoxytryptophol or of synthetic analogues (hexanoyl methoxytryptamine, propionyl methoxytryptophol, or 6-chloro-melatonin) in Exp. 1 or of a natural analogue (N-acetylserotonin, 6-hydroxy-melatonin, hydroxytryptophol, or methoxytryptophol) in Exp. 2 had no effect on the circulating T4 levels. Plasma levels of triiodothyronine (T3) and thyrotropin (TSH) were unaffected in either experiment. Since none of the tested melatonin analogues is capable of suppressing circulating T4 concentration when given in a dose at which melatonin is reproducibly effective, the pineal-induced suppression of T4 is most likely mediated by melatonin. Plasma cholesterol levels were elevated only in hamsters receiving 6-chloromelatonin injections. However, plasma triglyceride levels were significantly higher than the diluent treated controls in Exp. 1 after injections of melatonin, acetyl methoxytryptophol, propionyl methoxytryptophol and 6-chloromelatonin. Interscapular brown adipose tissue was significantly heavier in melatonin treated animals.

Influence of some pteridines on pineal 5-methoxy-indole synthesis in male wistar rats periodically exposed to either white or red light

In previous investigations the change of circadian rhythmicity in pineal melatonin/5-methoxytryptophol synthesis of rats periodically exposed to red light was similar to that in pineals of rats incubated with pterin-6-aldehyde. These experiments were, however, performed with rats of different age and in different periods of the year. In the present study these two factors influencing pineal indole metabolism have been combined the experiments being carried out in rats aged 28 days and during the same day in the month of January. It was observed that under influence of red light the peak of melatonin/5-methoxytryptophol synthesis shifted towards daytime, whereas incubation with pterin-6-aldehyde did not cause such a shift. If under different experimental conditions the mean amount of melatonin/5-methoxytryptophol which was formed over a 24 hour period was compared, it appeared that pineals of rats exposed to white light incubated with reduced neopterin but not pineals incubated with pterin-6-aldehyde behave in this respect similar to pineals of rats exposed to red light. However, if the ratio between melatonin/5-methoxytryptophol and 5-methoxytryptamine is calculated pineals of white light exposed rats incubated in pterin-6-aldehyde behaved very similar to the pineals of rats exposed to red light. Although the role of pteridines remains obscure, it appears that the parameters 2. circadian rhythmicity and 2. the amount of 5-methoxyindoles and 3. the ratio between these indole derivatives might be of importance in analyzing their physiological effects. The influence of application of light of different wavelengths and year rhythmicity is discussed.

Alterations in the spontaneous activity of cells in the guinea pig pineal gland and visual system produced by pineal indoles.

The indoles serotonin (SER), melatonin (MEL), 5-methoxytryptophol (5-MTL) and 5-hydroxytryptophol (5-HTL) were administered during daytime microelectrophoretically to 240 cells in the pineal gland of the guniea-pig. The action of SER and 5-HTL was predominantly depressant on the electrical activity, MEL and 5-MTL caused an excitation in most of the units. Although MEL and 5-MTL caused fairly similar reactions on average, they appear to act on different cells. The effects of microelectrophoretically applied MEL and 5-MTL on the spontaneous or evoked activity in the visual system (retinal ganglion cells, optic tract, lateral lateral geniculate body, superior colliculus) of the guinea-pig were also investigated. Of the 76 cells tested in the visual system 25 of the ON- and OFF-cells increased the rate of discharge when the two indoles were applied. Cells in the optic layer of the superior colliculus showed no measurable response to the application of the two substances. 5-HTL caused no effect on cells in the visual system.

The effect of melatonin and other indole derivatives in maintaining ovulation in rats kept in continuous light and the influence of these indoles on HIOMT activity in the pineal gland.

The aim of the present study was to investigate whether the ovulation-maintaining effect of melatonin in rats, exposed to continuous light (LL), was also exerted by other pineal indoles which have been reported to influence the reproductive processes of mammals. The effect of 10 micrograms melatonin was compared with that of similar amounts of either N-acetylserotonin, 5-methoxytryptophol, 5-methoxyindole-3-acetic acid, 5-hydroxytryptophol, 5-methoxytryptamine or 5-methoxytryptophan. All these compounds appeared to be significantly less effective than melatonin in preventing the effect of LL, ovulation being preserved in only 20--33% of the rats investigated, with melatonin this percentage being 60--75%. Investigations were also carried out to assess the effect of these indole derivatives on HIOMT (hydroxyindole-O-methyl transferase) activity in synthesizing different 5-methoxyindoles in the abnormally influenced pineal gland due to LL. Melatonin, the compound the effect of which on ovarian cyclicity is strongest, stimulates 5-methoxytryptophol synthesis; while other less active compounds stimulate the synthesis of melatonin and inhibit that of O-acetyl-5-methoxytryptophol. The possibility that the effect of other indoles than melatonin on ovarian cyclicity might be due to stimulation of melatonin synthesis was considered. A possible functional relationship of the different indoles cannot be excluded.

An in vitro stimulatory effect of indoleamines on aldosterone biosynthesis in the rat

The effect of the indoleamines, serotonin, 5-methoxy-tryptamine, 5-methoxytryptophol and melatonin on aldosterone biosynthesis by rat adrenals in vitro has been studied. In all experiments incremental doses of each of the indoleamines tested produced a progressive increase in aldosterone secretion. Serotonin (10 −6 mol/l) produced a 5-fold increase in aldosterone secretion by isolated perfused rat zona glomerulosa cells obtained from animals on a normal sodium diet compared to the basal levels. Higher (9-fold) responses were elicited when the cells were from sodium deficient rats and lower (3-fold) when the rats were sodium loaded. 5-methoxytryptamine (10 −6 mol/l) was a more potent aldosterone agonist (8-fold increase) and the aldosterone response was similarly affected by change in sodium intake. Smaller responses were found when cells were stimulated by 5-methoxytryptophol (10 −4 mol/1, 3.5-fold) or melatonin (10 −4 mol/l, 3-fold). The dopamine agonist serotonin antagonist Lisuride completely inhibited the aldosterone response to 5-methoxytryptamine (10 −8 mol/1) and serotonin (10 −7 mol/l). These results suggest that pineal indoles could play a role in aldosterone biosynthesis and indicate that changes in sodium balance affect the aldosterone response to indoles in the same way as has been described for other aldosterone secretagogues.

Effect of photoperiod and pineal indoles on the reproductive system of young female white-footed mice.

Exposure of prepubertal female mice to short photoperiod (LD 8:16) for 3 weeks resulted in a significant inhibition of reproductive tract growth compared with mice maintained in long photoperiod (LD 16:8). Chronic subcutaneous melatonin-beeswax implants containing 1 mg melatonin also produced an inhibition of the reproductive system of these animals. The repressive effects of melatonin appear not to be influenced by photoperiod since comparable results were obtained from groups of mice bearing implants in long and short photoperiods. In addition, the ability of beeswax implants containing various indolic compounds (melatonin, serotonin, 5-methoxytryptamine, 5-methoxyindole-3-acetic acid, and N-acetylserotonin) to inhibit the growth of the reproductive system of this species were compared. Only implants containing melatonin or serotonin caused any significant repression of uterine and ovarian weights.

Selectivity of melatonin pituitary inhibition for luteinizing hormone-releasing hormone.

The pineal indole melatonin suppresses the neonatal rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responses to LH-releasing hormone (LHRH), as shown in previous studies from this laboratory. We show in this study that the melatonin inhibition is a selective effect and is not due to general inhibition of pituitary function. The effects of the indole on the responses to thyrotropin-releasing hormone (TRH) and somatostatin (SRIF) and on basal pituitary hormone secretion were examined with cells in culture. Neonatal rat anterior pituitary cells dissociated with collagenase and hyaluronidase were cultured overnight and distributed to 35-mm dishes at the time of use. For examination of melatonin effects on the response to releasing hormones, the cells were incubated for 3 h in control medium or medium containing LHRH (10-9-10-6 M), TRH (10-10-10-6 M), or SRIF (10-9-10-6 M), either alone or in the presence of melatonin (10-8 or 10-6 M). For examination of basal hormone secretion, the cells were incubated for 1.5, 3, 6, 15, or 24 h in either medium alone or medium containing melatonin (10-6 M). Medium and cell lysate concentrations of LH, FSH, thyroid-stimulating hormone (TSh), prolactin (PRL) and growth hormone (GH) were determined by double antibody RIA. As previously, melatonin (10-8 M) significantly suppressed LH and FSH release by all concentrations of LHRH. This concentration of the indole produced maximal suppression of both LH and FSH responses to LHRH. By contrast, melatonin at a 100-fold greater concentration (10-6 M) had no effect on TRH stimulation of TSH or PRL release or on SRIF inhibition of GH release. Similarly, melatonin had no effect on basal release of TSH, PRL, or GH at the times examined. These findings show that melatonin inhibition of the gonadotroph response to LHRH is a selective effect.

REM sleep suppression in cats by melatonin

The pineal indole melatonin, injected into the third ventricle of unanesthetized cats, induced NREM sleep within 11–15 min and suppressed REM sleep for about 3 hr. A dose-related effect could be established between 1, 10 and 100 ng melatonin. The reappearance of REM sleep at about 3 hr following melatonin administration occurred with a marked rebound. The present results further support the hypothesis that melatonin represents the releasing hormone for pineal arginine vasotocin.

Changes in the rat sleep-wake cycle produced by DL-6-fluorotryptophan, a competitive inhibitor of tryptophan hydroxylase.

DL-6-Fluorotryptophan (6-FT), a competitive inhibitor of tryptophan hydroxylase, produced a transient disruption of sleep in rats chronically implanted with EEG recording electrodes. In the 4 h period following the administration of 6-FT (120 mg/kg) awake time was increased, paradoxical sleep time was decreased and slow-wave sleep remained unchanged. These sleep changes were accompanied by significant reductions in brain 5-HT levels. L-Tryptophan (100 mg/kg) co-administration with 6-FT prevented the major sleep changes whereas L-leucine (100 mg/kg) was without effect. The major sleep changes produced by 6-FT were prevented by the pineal indole melatonin (20 mg/kg) but not by L-5-hydroxytryptophan (5 mg/kg). These neurochemical and drug interaction data raise the possibility that 5-hydroxytryptamine is involved in the control of paradoxical rather than slow-wave sleep in the rat.

The role of the pineal gland in neuroendocrine control mechanisms of neoplastic growth.

A survey of papers read during a workshop held in Vienna, 1977, is given showing that the pineal gland can be considered a model for research on neuroendocrine control mechanisms in neoplastic growth. From data obtained by different authors it can be concluded that the role of the pineal indole melatonin is very important in regard of the incidence and the development of tumours. On the basis of all the facts mentioned in this paper it is obvious that pineal gland activity should be included into the neuroendocrine factors studied in relation with oncogenic processes. For future research a number of experimental approaches, which need special emphasis, are recommended.

Vasotocin, melatonin and narcolepsy: Possible involvement of the pineal gland in its patho-physiological mechanism

Both the pineal nonapeptide hormone arginine vasotocin (AVT) (2.5 μg) administered intra-nasally and the pineal indole melatonin (50 mg) administered intravenously to three male narcoleptics (two with auxiliary symptoms and one with sleep attacks only), dramatically increased the amount of REM sleep and decreased REM sleep latency. The duration of the sleep onset REM periods in the two narcoleptics with auxiliary symptoms increased by more than 100 percent after AVT and melatonin administration. In the narcoleptic with sleep attacks only both AVT and melatonin induced REM periods at sleep onset. The hypothesis is advanced that narcolepsy represents an impairment of the melatonin-AVT control in the induction and circadian organization of REM sleep associated with an immaturity of REM triggering centers.

Melatonin blocks in vitro generation of prostaglandin by the uterus and hypothalamus

The effects of melatonin on the motility (isometric tension developed and contractile frequency) of uterine horns isolated from ovariectomized rats as well as on the mechanical responsiveness to added oxytocin or prostaglandin F 2α (PGF 2α) were explored. The pineal indole (10 −6 or higher) depressed significantly the spontaneous motility of the uterus and reduced the responses evoked by oxytocin but not those evoked by PGF 2α. Melatonin was also tested on the prostaglandin (PG) release into the suspending media from either uterine horns from spayed rats or bovine medial basal hypothalamic (MBH) explants. Melatonin (10 −3 M) diminished the output from the uterus or the MNH of both PGE and PGF-‘like material’. Similarities between the effects of melatonin and indomethacin as well as the possible physiological relevance of the present findings are discussed.

Melatonin inhibition of the in vivo pituitary response to luteinizing hormone-releasing hormone in the neonatal rat.

The effects of melatonin on the in vivo pituitary LH response to LH-releasing hormone (LHRH) were examined in neonatal male and female rats, in 35- to 44-day-old male rats, and in 35- to 44-day-old male animals which had been either pinealectomized or maintained in constant light for at least 3 weeks before use. Animals were given saline or melatonin (1--100 micrograms/rat) followed within 30 sec by saline or LHRH (10--1,000 ng/rat) at separate subcutaneous sites. Blood was collected following decapitation either without prior injection or 15, 30, 45, or 60 min afterwards. Serum LH concentrations were determined by double antibody radioimmunoassay. In neonatal male and female rats, melatonin (1 microgram) significantly (p less than 0.01) suppressed by approximately 65% serum LH at 15 min after LHRH. Suppression was maintained for at least 60 min, a finding which indicates that melatonin blocks rather than delays the response to LHRH. By contrast, in normal, pinealectomized, and constant light older male rats, melatonin (100 micrograms) had no detectable effect on either the magnitude or the time course of LH release by LHRH. These data extend our previous in vitro findings by demonstrating that melatonin is a potent inhibitor of the in vivo pituitary response to LHRH in neonatal rats but not in older animals. Neither pinealectomy nor constant light, both of which are assumed to reduce pineal melatonin production, for at least 3 weeks before use restores neonatal pituitary responsiveness to the pineal indole in the older animals.

Immediate response of 5-methoxytryptophol in the circulation to hypoglycaemic stress induced by insulin.

Pineal indoles have been shown to affect the release of anterior pituitary hormones but details of the interrelationships are lacking. Using a new gas chromatography-mass spectrometry (g.c.-m.s.) assay the concentration of 5-methoxytryptophol (ML) was measured in plasma samples obtained from 16 children undergoing investigation of pituitary function for delayed growth. All the children received an insulin tolerance test (ITT) to study their endocrine response to stress. Some children received luteinizing hormone releasing hormone (LH-RH) and/or thyrotrophin releasing hormone (TRH). The change in concentration of ML during an ITT was similar to the change in concentration of blood sugar; a drop at 20 min followed by a rise at 30 min. This was not significantly altered by the administration of LH-RH or TRH, nor was there a different pattern of response in children who were deficient in growth hormone as opposed to those with idiopathic delayed growth. The fall in concentration of ML with stress may mediate the increased secretion of pituitary hormones. Alternatively, the pineal gland may respond directly to insulin.

Melatonin, the pineal gland and human puberty.

Animal experiments have suggested that the pineal gland produces an anti-gonadotropic hormone. The hamster, for example, undergoes reproductive collapse when kept in short-day periods, an effect which is abolished by pinealectomy. Although there is little direct evidence about the endocrine role of the pineal gland in man, it has been noted that tumours of the pineal gland in young boys are associated with precocious puberty and the human pineal gland has been suggested to produce a substance that holds sexual maturation in check. This observation has been extended by Kitay, who has shown that destructive tumours are associated with precocious puberty whereas hyperactive tumours are associated with delayed puberty. However, no studies have described any change of pineal function with normal puberty. Because two pineal indoles, melatonin and methoxytryptophol, have been shown to be antigonadotropic when administered to animals, we have now measured them in schoolchildren. Our findings show that in young boys there is an abrupt fall in the concentration of melatonin with advancing development suggesting that it may play an important physiological role in the control of human puberty.

A sensitive and specific assay for 5-methoxytryptophol in plasma.

5-Methoxytryptophol (ML) is found in the pineal gland and is known to have biological activity especially as an antigonadotrophic agent, but methods have been lacking for its measurement in the circulation. A gas chromatography-mass spectrometry assay using a trimethylsilyl derivative has been developed for the routine measurement of ML in plasma. The assay is of great specificity and has a sensitivity of 20 pmol/l. Studies on the levels of pineal indoles in the circulation, however, have been hampered by the possibility that extraneous compounds are being cross-measured. Thus the specificity of the routine assay has been further validated by comparing it with an alternative assay system where all the major parameters were changed, i.e. derivatizing reagent, internal standard and mass number. Results that were obtained using both assay systems were closely comparable.

Changes in the concentration of 5-methoxytryptophol in the circulation at different phases of the human menstrual cycle.

The pineal indole 5-methoxytryptophol (ML) has been shown to have an antigonadal activity when administered to experimental animals, but data on its normal pattern of secretion have been lacking. Using a new gas chromatography-mass spectrometry assay, the concentration of ML at various phases of the human menstrual cycle has been studied. Daily samples were obtained throughout the month from five women with a normal cycle and two women taking an oral contraceptive. In women with a normal cycle levels of ML were found to be significantly lower in the last third of their cycle; this change was not seen in women taking an oral contraceptive who had low levels throughout the month. The changes in concentration of ML did not correlate with the changes in concentration of gonadotrophins.