Pineal indoles: Significance and measurement

Neuroscience &BiobehavioralReviews,Vol. 10, pp. 273--293,1986. ©AnkhoInternational Inc. Printed in the U.S.A.

0149-7634/86$3.00 + .00

Pineal Indoles: Significance and Measurement P A U L B. F O L E Y , K E I T H D. C A I R N C R O S S 1 A N D A N D R E W F O L D E S *

School of Biological Sciences, Macquarie University, North Ryde, N.S.W. 2113, Australia and *C.S.I.R.O. Division of Animal Production, P.O. Box 239, Blacktown, N.S.W. 2148, Australia R e c e i v e d 3 J a n u a r y 1986 FOLEY, P. B., K. D. CAIRNCROSS AND A. FOLDES. Pineal indoles: Significance and measurement. NEUROSCI BIOBEHAV REV 10(3) 273-293, 1986.--Despite intensive investigation, particularly over the past fifteen years, many aspects of pineal function with respect to mammalian physiology remain obscure. Much of this work is reviewed and particular attention focussed on indole metabolism within the pineal gland. Emphasis is placed on the development of new analytical techniques with especial reference to high performance liquid chromatography coupled with electrochemical detection. The growth in knowledge regarding pineal indole synthesis which can be attributed to the use of this technique is discussed. The possibility that pineal indoles other than melatonin may function as hormones or neuromodulators is considered. A functional role for 5-hydroxytryptophol as a neuromodulator, possibly associated with diffuse neuroendocrine function (amine precursor, uptake and decarboxylation, APUD) is suggested. Pineal function

High performance liquid chromatography

Mammalian physiology

as well as further inputs from central structures including the hypothalamus and amygdala [73, 74, 147,166, 217,218,227, 272]. Recent microscopic investigations afford support for the importance of central innervation in pineal function, establishing the presence of large numbers of unmyelinated afferent and efferent fibres in the pineal stalk of the rat [50, 75, 129, 228]. The pineal is unique among endocrine organs for a number of other reasons: (1) it is one of the few unpaired endocrine organs; (2) on a weight basis, it receives one of the richest blood supplies of any organ; (3) it lies outside the blood brain barrier, but has direct access to cerebrospinal fluid (CSF) via the third ventricle (though such access in most rodents has been disputed [107]). It also has indirect access via a branch of the cerebral artery passing to the choroid plexus of the third ventricle [107,205]. It is in contact with, and may form a functional unit with the subcommissural organ [75,76]; (4) it produces and/or contains high concentrations of a number of different indoleamines and low molecular weight peptides of probable endocrine importance; (5) it is responsive to changes in magnetic field strength and to external electrical stimuli (see, for example [127, 231,233,280, 290, 302]. The significance of such sensitivity requires further study. Current pineal research appears to be concentrated on three aspects: (a) the role of the pineal in reproduction and other photoperiodic phenomena; some, such as pelage changes, being of potential commercial importance; (b) the precise rhythmic regulation of the synthesis and release of the best characterised pineal indoleamine, melatonin and (c) the role of the gland in human physiology in health and disease.

ASPECTS OF PINEAL FUNCTION Despite intensive investigation, especially over the last fifteen years, much still remains to be understood concerning the function of the mammalian epiphysis, or pineal gland. Revered since antiquity for its speculated association with the soul, in our century the gland has been linked primarily with the photoperiodie regulation of reproduction, and considered to be a non-functional vestigial organ in man. Although in recent years our knowledge of the pineal has grown at a rapid rate, it has become increasingly difficult to define the scope of pineal action. It is becoming evident, however, that the narrow role currently ascribed the gland reflects only a small part of its overall function. The breadth of its suspected influence was concisely expressed by Ariens Kappers in 1981 [122]: "the (mammalian) pineal organ i s . . . an endocrine organ of neural origin being of multifunctional significance in modulating the function of endocrine, and probably also of nonendocrine regulatory systems, synchronizing this function with internal and external conditions." In other words the pineal functions as the regulator of regulators, linking external conditions with the "milieu interhale." A schematic diagram of the flow of information into and out of the mammalian pineal organ is shown in Fig. 1. It is now becoming accepted that, apart from relatively well characterized responses to photic stimuli, the pineal also receives olfactory, electromagnetic and acoustic inputs, 1Requests for reprints should be addressed to Keith D. Cairncross.

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Reproduction The involvement of the pineal gland in reproductive phenomena was the first suggested, and the most studied of the many possible endocrine roles of the gland (see, for example [133]). Experiments involving pinealectomy and/or melatonin administration in different breeds of hamsters, performed under different conditions, led to conflicting hypotheses regarding the roles of the pineal in reproduction. For example, Carter et al. [58] claimed the gland to have solely progonadal function, whereas Reiter [209] suggested an antigonadal effect for the gland. The one consistent finding is that in both short and long day breeders, a clear interaction between photoperiod and the reproductive system involving the pineal gland has been demonstrated. In the sheep, a short day breeder, pinealectomy [38], ganglionectomy [154,155] and melatonin supplementation [128] experiments have shown involvement of the gland in the response of the reproductive system to changes in photoperiod. Bittman et al. [37] have further shown that the effects of pinealectomy on LH secretion can be reversed by appropriately timed melatonin infusions, suggesting that melatonin is the pineal principle involved in mediating the reproductive effects in the ewe. The duration, phasing and amplitude of the nightly melatonin peak may all be important means of signalling seasonal information to the hypothalamopituitary-gonadal axis in different species [259]. These authors summarize the role of the pineal in reproduction in the sheep as follows: "The results o f . . . experiments in different species suggests neither a pro- nor an antigonadal role for the pineal and its melatonin. Rather, they indicate that in order to correctly interpret day length, animals must experience a daily rhythm in melatonin. The almost equivalent effects of pinealectomy, ganglionectomy and melatonin implants on seasonal reproductive cycles stem from the fact that these procedures eliminate transduction of the external lighting cycle. °' A seasonal gonadal steroid-mediated feedback regulation of

pineal function has been suggested in sheep [96,98] but the mechanisms by which the pineal refers photoperiodic influences to the reproductive system remain to be fully elucidated. In humans the study of pineal involvement in reproduction has been mostly confined to regulation of the onset of puberty, with inconsistent results [258]. Pelage Change in Sheep Melatonin treatment or pinealectomy have been shown to cause contrasting changes in coat colour or thickness in a variety of species, including the hamster [112], the mouse [160], and deer [201]. In sheep, recent work on seasonally shedding breeds suggests that pinealectomy [289] and superior cervical ganglionectomy [155] bring about significant alterations in the timing of the photoperiod-controlled seasonal shedding cycle. Further studies are needed in this area to elucidate the endocrine mechanisms involved in pineal regulation of wool and hair growth. Studies on the Regulation of Melatonin Synthesis and Release Whereas studies on reproductive and pelage-related effects of the pineal focus on longer term, seasonal rhythms, studies on melatonin synthesis and release focus on circadian rhythms, that is events recurring with a frequency of approximately 24 hours, which are studied under natural or artificial light conditions, and which may be entrained to environmental stimuli or zeitgebers. For further information on these much studied aspects of pineal function, the reader is referred to recent reviews [32, 89, 204]. The Role of the Pineal in Human Physiology In humans, as in other species, melatonin is the most studied pineal indole, and its regulation and synthesis appear to be analogous to that observed in other species, except that much higher light intensities are required to suppress its secretion [152], leading to its secretion throughout the day in the presence of artificial lighting [279] and less marked sea-

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sonal differences [120]. In humans, melatonin is released into the systemic circulation, where it binds to plasma proteins, and is taken up by a range of tissues. The major urinary metabolite in man is sulfate-conjugated 6-hydroxymelatonin [243], while a small amount of unchanged melatonin is also excreted [161]. A significant proportion is also excreted as N-acetyl serotonin, the precursor of melatonin [296]. As in other species there is a growing body of evidence for melatonin involvement in aspects of human reproduction [34] including pregnancy, fetal development and parturition [172], and possibly in regulating the onset of puberty ([133,275]; but gee also [87]). In addition, the pineal also appears to influence a range of human endocrine organs and central neuronal mechanisms [212]. These include: (1) Regulation and induction of noctur-

hal sleep [36]. Melatonin changed EEG activity in healthy adult males; peaks and troughs in endogenous melatonin secretion correlated with waking episodes and REM sleep respectively. This seems to be contradictory to the reported tranquilizing and sleep inducing effects of exogenous melatonin [7, 35, 69, 225,292]. (2) Thyroid hormone secretion [274]. This effect of melatonin may be mediated via action on hypothalamic TRH release. Conversely, exogenous TRH has been reported to stimulate melatonin synthesis in healthy humans [34]. (3) Growth hormone activity has also been reported to be influenced by melatonin [8, 245, 246] but no definitive effects on human growth have yet been reported [35]. Changes in melatonin rhythms have been shown at different stages of the menstrual cycle with different seasons [144] and also with age [83,116].

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FIG. 3. Simplified model of neural control of the pineal gland. The broken line indicates a hypothetical sequence (adapted from [55,135]). TRP=tryptophan, 5HTP=5-hydroxytryptophan, 5HT=5-hydroxytryptamine, 5HTL=5-hydroxytryptophol, 5HIAA=5-hydroxyindoleaceticacid, NAS=N-acetylserotonin, MEL= melatonin, 5MTL=5-methoxytryptophol,5MIAA=5-methoxyindoleaceticacid, PG=prostaglandin. Other abbreviations as in Fig. 2. Although "definitive evidence for etiological involvement of melatonin has not been demonstrated in human medicine" [35], changes in melatonin rhythm have been associated with a large number of pathological states [35], and with the mode of action of therapeutic agents used to treat widely differing symptoms [34]. Changes in aspects of pineal function [20,258] and/or effects of exogenous melatonin have been reported in patients with malignant tumours/carcinomas [46], while exogenous melatonin has been shown to inhibit tumour induction [143,207]. Conversely, Cohen et al. [65] have hypothesised that the pineal is an initiating factor in certain forms of carcinogenesis. Such a view receives support from studies implicating the pineal gland as a controlling factor in the mammalian immune system [84,261]. More recently, melatonin deficiency has been linked with a variety of disorders including depression, peptic ulcer and sexual dysfunction [165]. In view of the tranquilizing effects of exogenous melatonin [292], it has been used in the treatment of depression, schizophrenia and Parkinson's disease, with limited success [57,151]. However, both endogenous melatonin levels, and the ratio of melatonin to cortisol appear to be clinically useful markers for affective psychiatric disease [283], despite the lack of significant interaction between melatonin and cortisol in healthy volunteers [8]. Considering the suggested widespread effects of melatonin on the endocrine and APUD [193] systems, future studies in man should result in further clinical applications for melatonin, and also for the other pineal indoles for which no clinical functions are yet firmly established, to overcome or counteract a range of syndromes brought about by endocrine imbalances.

An interesting application of exogenous melatonin, which is currently receiving attention, is the possibility of correcting phase shifts in circadian body rhythms such as those experienced by shift workers and long distance air travellers [93]. The next few years should bring about large advances in our knowledge and ability to overcome the deleterious effects of such phase shifts. PINEAL INDOLE METABOLISM The key to understanding the physiological significance of the pineal lies in precise knowledge of the substances it manufactures and releases, and of the factors which control these processes, including the enzymic systems implicated in such functions. The pineal contains all necessary enzymes required for energy production, as well as for peptide and indole synthesis [205], but since the identification of melatonin in the pineal [150], the focus of attention has been on indole metabolism (see [ll9, 139, 204, 210]). Pineal indole metabolism may be divided into three major pathways (Fig. 2). The conversion of tryptophan to serotonin (5-hydroxytryptamine) is common to all three pathways. The first step in the biosynthesis is catalyzed by the rate limiting enzyme tryptophan hydroxylase (TH). Pineal mitochondria contain high concentrations of TH [205]; the enzyme is regulated by extracellular, and ultimately by circulating free, tryptophan levels [135,240, 241,295] and may exhibit circadian rhythmicity [238]. The second enzyme in the synthesis of serotonin is aromatic amino acid decarboxylase (AAAD), a cytoplasmic enzyme of low substrate specificity which may be sensitive to prolonged darkness [135]. Although the enzymic steps leading from serotonin to synthesis of the

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FIG. 4. NAT-activity related events occurring in the pineal gland during a 12 hour light-12 hour dark photoperiod (adapted from [31]). pineal hormone melatonin were rapidly elucidated following the isolation of melatonin in 1958, the complexity of the intricate regulatory system involved is only now beginning to be appreciated. A simplified model of the operation of this pathway is shown in Fig. 3. The pathway is confined largely to the pineal gland in mammals, but has also been reported in several species in the retina, Harderian gland and intestine [10, 17, 30, 135, 204, 288]. SEROTONIN N-ACETYL TRANSFERASE

The first step in the pathway, N-acetylation of serotonin by serotonin N-acetyltransferase (NAT) is generally, though not universally [71], regarded as the rate-limiting step. NAT is distinguished from acetyltransferases in other tissues by the endogenously generated large amplitude variations in its activity (at least 3 times higher at night than during the day in sheep [177], 30--100 times higher in rats and other species; it has been suggested that considerably different mechanisms control melatonin synthesis in rats and in species with low amplitude NAT rhythms, such as the sheep [177], and that, in fact, such synthesis is regulated in the ovine pineal by a NAT-independent process [170]), entrained to variations in environmental lighting. NAT is an enzyme of about 39,000 daltons molecular weight, and transfers an acetyl group from its co-factor, acetyl Coenzyme A, to an amine acceptor. This acceptor is usually serotonin, but other pineal indoleamines (5-methoxytryptamine and tryptamine) are equally good substrates [78]. NAT is subject to complex regulation by several interacting mechanisms (Fig. 4). Intracellular Mechanisms

NAT activity is unstable in vitro unless maintained at pH

6.8 in the presence of acetyl CoA or, less effectively, CoA, cystamine, penicillamine or/3-mercapto-ethylamine; as there are high concentrations of compounds related to sulfur metabolism in the pineal [31], NAT may be stabilized in vivo by conversion to a thioacetyl form [33, 134, 135]. The decrease in NAT activity on exposure to-light is very rapid (tl/2=/l min) and resembles that of the unprotected enzyme in vitro [178], so that this may be a physiological mechanism. Alternatively, acetyl CoA may compete for binding to NAT with an inhibitor, " N A T inhibiting substance" or NIS. It is not yet known whether this selective inhibitor actually binds NAT, or whether it is a protease with which NAT synthesis competes: NIS is known to be unique to the pineal, and is not influenced by norepinephrine [31, 62, 84, 134]. NAT activity is initiated by stimulation of the adrenergic-cAMP system (see below); mRNA synthesis is required for induction of the enzyme initially, but after this has been accomplished, protein synthesis is the only requirement for elevated NAT activity [31, 61, 135, 137, 176, 298]. The protein synthesized may not be NAT itself, but perhaps some activating or inhibiting substance. Cyclic AMP has a threefold role to play in this regulation. Firstly, it induces phosphorylation of nuclear proteins promoting mRNA synthesis for NAT and initiator manufacture [32]. Secondly, cAMP may facilitate the interaction between NAT and acetyl CoA molecules, competitively displacing inhibitor (NIS) molecules which suppress NAT activity in the absence of cAMP [ 134]. Finally, the rapid decrease in NAT activity at the end of the dark period is preceded by an even more precipitous decrease in cAMP; this decline in the "second messenger" may be the signal for the inactivation of NAT [138]. cAMP may act via membrane hyperpolarization, causing an altered redox status of the cytosol, with consequent effects on (in)activating substances within it [134].

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TABLE 1 DRUGMODULATIONOF NAT ACTIVITYOR METABOLISM Dru~s Which Stimulate NAT Activity adrenergic agonists

norepinephrine, epinephrine, isoproterenol, L-DOPA, phenylephrine, terbutaline, dobutamine. cAMP mimics and phosphodiesterase inhibitors

dibutyryl cAMP, PGE2, VIP, cholaragen, isobutylmethylxanthine, theophylline. monoamine oxidase inhibitors

Catron, pheniphraxine, pargyline, harmine. agents which affect neurotransmitter functions

desmethylimipramine, tyramine, cocaine, KCI depletion, chlorpromazine, veratridine. Drugs Which Inhibit NAT Activity [3-adrenergic blocking agents

propranolol. protein synthesis inhibitors

cycloheximide, 2-fluorohistidine. RNA synthesis inhibitors

actinomycin D, ~-amanitin, cordyeepin. sulfur group-inactivation

cystamine, ethylmaleimide. prostaglandin synthesis inhibitors

indomethacin substrate inhibition

serotonin cholinergic blocking agents (acting via SCN)

carbachol, atropine miscellaneous agents

KCI, reserpine, tetrodotoxin, nicotine, anaesthetic barbiturates, morphine, lidocaine. modified from [31].

Norepinephrine (NE) binds to/31-adrenergic receptors in pinealocyte membranes, and initiates a chain of events beginning with enhanced cAMP synthesis and leading eventually to increased NAT activity (see above). Regulation of the rapid NAT decline is less well understood, but may relate to a-adrenergic receptors located in the central nervous system [2]. This complex noradrenergic regulation of NAT appears to be unique to the pineal gland [45, 79, 80, 119, 298]. Other compounds also play a role in the pineal synapse. NE, as well as binding to /31-adrenoceptors, also binds to a-adrenoceptors in the pinealocyte membrane, accelerating phosphatidylinositol turnover [27,299]. This results in the intracellular release of a number of secondary messengers, including diacylglycerol, inositol triphosphate and phosphatidic acid, many of whose effects [28] may explain the potentiation of pineal/3-stimulation by a-adrenoceptors [36, 254, 256, 266]. These effects include the release of intracellular stores of calcium and the activation of protein kinase C [255] and the augmented synthesis of arachidonic acid, the precursor for prostaglandins E2 and F2~, which bind specific pinealocyte receptors [52-55, 221, 222]. Both these effects enhance cAMP accumulation and NAT activity [222,255]. Additionally, Vacas et al. [264] describe a-potentiation of/3-adrenergic depression of phosphodiesterase activity. Parkington et al. [192] have similarly described interaction between a- and /3-induced electrical events in the pineal gland. cGMP synthesis in the pineal is especially dependent on a-stimulation [266]; arachidonic acid is known to facilitate guanyl cyclase activity [28]. Similar interaction between a- and/3-adrenergic systems have been reported in the limbic system [39] and in rat striatal [145] and cortical [200] slices, and direct interreceptor modulation cannot be rejected; this is not a novel concept [ 140,162]. Gamma-aminobutyric acid (GABA) uptake and synthesis has been demonstrated in the pineal gland. In bovine [62] and ovine [97] pineal glands, GABA, possibly acting via cGMP, inhibits NE-stimulated, but not basal, N A T activity. In the rat, no such effect was noted [163], but this may be dependent on the phase of the light/dark cycle, or co-factor availability [15,17]. In the ovine pineal the locus of action of GABA on NAT activation is distal to the action of cAMP [97] and may be at the level of inhibition of protein kinase

PINEAL INDOLES activity. When the role of GABA in the pineal is elucidated, it may be proved to be the first exception to the hitherto observed absolute adrenergic specificity of the NAT regulatory system [17]; it may also be important in the O-acetyltransferase system [15]. Finally, a recent report suggests that vasoactive intestinal polypeptide (VIP, a putative neuromodulator with acetylcholine in some cholinergic neurons [158], which may have some neurotransmitter-like properties of its own [90]) enhances the stimulatory effect of NE on NAT activity by means as yet unknown [297]. Neural Circuitry The suprachiasmatic nucleus (SCN) primes the pinealocytes for activation at about 12-hourly intervals, the endogenous rhythm being entrained to environmental lighting. Transmission of signals from the SCN is blocked by light; thus the rhythm persists in darkness, but is suppressed by constant light. The cycle is conveyed to the pineal gland via both central and peripheral structures (Fig. 5) [74, 75; 133, 139, 147, 204, 217]. The clock is highly stable; NAT activity will not increase during the daytime even if animals are placed in darkness, and conversely, NAT activity will decline on schedule, even in darkness, producing a peak of shorter duration than usual, following extension of the light period [134, 135,262, 301]. Pharmacological Agents A partial list of the various classes of pharmacological agents which affect NAT activity is shown in Table 1. In addition, receptors for benzodiazepines, a class of potent centrally-acting anxiolytic drugs, have been found in the pineal gland [ 157]. Benzodiazepines appear to augment NAT activity [164], whereas antidepressants such as imipramine depress it [101]. These opposing effects of the two classes of drugs may be expected on the basis of their differing sites of action, benzodiazepines having been suggested to compete with GABA, and imipramine acting as an inhibitor for neuronal reuptake of NE into presynaptic nerve terminals, as well as antagonising the ct2 adrenoceptor. Miscellaneous Agents Taurine. Taurine has been claimed to be released from presynaptic nerve terminals with NE; its release appears to be achieved by a /3-adrenergic mechanism [286]. Taurine binds /3-receptors with less potency (for NAT induction) than NE and decreases the potency of NE to induce NAT [ 10]. Taurine may also feed back on the terminal to inhibit NE release [285]. Lighting. NAT suppression by light intensity follows a dose-response relationship in a number of species [30, 40, 277] but the threshold intensity is much higher (0.005-0.019 t~W/cm2) than that required for visual function (10-6 /xW/cm 2) and is not constant, being dependent on species and, within species, on previous photoperiod experience [277]. After a normal 12 hour light period, NAT rises for about 12 hours in the dark, but will be rapidly suppressed by as little as 60 seconds of light exposure. Depending on the stage of the dark cycle at which the animals are exposed to light, NAT activity will recover later in the same night, or not until the following dark cycle [32, 117, 118,265]; see also [286,287]. Acute exogenous stimuli. Pineal NAT activity can be

279 suppressed by short term exogenous stimuli, including immobilization, swimming in cold or warm water, and exposure to heat, noise or hunger [272,281] albeit at supraphysiological levels only. Despite increased catecholamine secretion due to stress, pineal NAT activity is not increased due to the efficiency of the presynaptic catecholamine reuptake mechanisms [134,190]. HYDROXYINDOLEO-METHYLTRANSFERASE(HIOMT) HIOMT catalyzes the O-methylation of 5-hydroxyindoles by the methyl donor S-adenosyl methionine (SAM). The enzyme has two 39,000 dalton subunits [135] and is present in the pineal gland in high concentrations, representing 2-4% of the total soluble protein. Similarly to NAT, HIOMT is also subject to different endogenous regulatory mechanisms. Intracellular Mechanisms The demethylated product of SAM, S-adenosyl homocysteine (SAH), is a potent inhibitor of methyltransferases. In the pineal, SAM is known to exhibit a circadian rhythm with a daytime peak in concentration [60]. Additionally, pyridoxal phosphate (Vitamin B6), which forms a Schiff's base with SAM, and thus inhibits the transfer of the methyl group, is also present in the pineal gland, as a co-factor in the biosyntheses of NE, taurine, GABA and 5H'F [10,186]. The existence of an endogenous disulfide inactivator has also been hypothesized [252]. Neural Circuitry Although regulated independently of NAT (HIOMT responds to changes in lighting over a period of days rather than minutes), HIOMT shares a common neural circuitry with NAT, including apparently the same/3-receptor-cAMP system [67] though dibutyryl cAMP cannot replicate this effect on HIOMT [30, 119, 135, 251]. Parallel changes in the response of HIOMT and cGMP to various photoperiods suggest that the two may be functionallyrelated in the pineal [139,250]. A seven day rhythm in HIOMT activity has been documented [273] but not verified independently of laboratory conditions; similar rhythms have been established previously for other endocrine functions [108]. Circulating Steroid Hormones In contrast to NAT, HIOMT activity appears to be highly responsive to gonadal steroids, though the evidence tends to be contradictory [56]. With the known involvement of melatonin in reproduction, some gonadal feedback on pineal melatonin biosynthesis would be expected. Foldes et al. [96,98] have shown a seasonal gonadal steroid-dependent feedback on pineal /3-adrenoceptors, which may reflect changes in the biosynthetic enzymes. Cardinali [51] found a dose-dependent enhancement of HIOMT activity by 17/3estradiol, by gonadotropins, and by testosterone at low concentrations, and suppression by high concentrations of testosterone and progesterone. On the other hand, other laboratories [124,250] found no evidence of any gonadal influence on HIOMT in studies involving adrenalectomy or hypophysectomy. Karasek and Reiter [123] and Fiske et al. [95] have found evidence of pineal-gonad-pituitary interaction, though whether the modification of HIOMT activity is due to steroids directly, or to changes in gonadotropin levels, re-

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O-ACETYL 5 METHOXY TRYPTOPHOL

ATONIN

FIG. 6. An alternative pathway of indole metabolism (after [13]). Solid line arrows: accepted melatonin biosynthetic pathway, broken line arrows: proposed alternative pathway to melatonin and other 5-methoxyindoles.

TABLE 2 PINEALOCYTE CELL TYPES Type 1 Activated by optic stimuli to eyes: subclassified on, off and on-off cells. Type 2 Exhibit transient bursts of electrical activity in response to acoustic input. Type 3 Exhibit biphasic response to stimulation of olfactory bulb. Type 4 Exhibit high level of spontaneous activity, abolished by superior cervical ganglionectomy. Type 5 Not described, but sympathetically innervated group in cells. See [234]. The first three groups are apparently innervated via the habenular and posterior commissures, the last two via the superior cervical ganglia.

mains to be established. Estradiol and other estrogens compete with NE for binding to/3-adrenoceptors in rat [51] and ovine pineal glands [98]. Pteridines

Rat pineal glands are rich in pteridines as a co-factor to tryptophan hydroxylase [205], and pteridines appear to regulate H I O M T activity in response to wavelength rather than intensity of incident light. Relative to white light, red light moves the peak in HIOMT activity to an earlier phase, and green light to a later phase [11]. Retinal extracts, containing endogenous pteridines, suppress H I O M T activity; synthetic pteridines vary in their ability to do the same [205]. Cremer-Bartels [70] suggested that there may exist a number of HIOMT isozymes, differing in pI, and that their relative concentrations in the pineal may depend on environmental lighting. The theory that HIOMT is a series of isozymes, independently manipulable by pteridines [12, 13, 17, 199], is now widely suggested [169,204]. This control was noted to be seasonal in the golden hamster [17] and to be age

dependent [ 14,18]. The discovery of this retinal-independent methylation has led to a suggested alternative biosynthetic route to melatonin (Fig. 6; [13]); this pathway appears to be suggested also by recent results in the ovine pineal [ 170,177]. Brainard et al. [41] similarly found differences in the ability of different wavelengths of light to suppress HIOMT activity, blue light (435-500 nm) being most effective. In man, serum folate concentrations fall during light adaptation, 6-formylpterin being a major metabolite; the rich blood supply of the pineal gland would allow such a photolytically produced pteridine to exert non-retinal control over HIOMT activity depending on incident light [71]. CONTROL OF THE MELATONIN BIOSYNTHETIC PATHWAY The question of whether N A T or HIOMT is the pacemaker enzyme is rather semantic. Although a large amplitude rhythm for HIOMT has recently been reported [66,67], it is generally agreed that the nocturnal HIOMT rise is insufficient to account for the rise in melatonin. HIOMT determines the maximum nocturnal output, but N A T de-

PINEAL INDOLES

281

TABLE 3 CHARACTERISTICSOF APUDCELLS--RELEVANCETO PINEALOCYTES Apud Cell Characteristic (1) (2) (3) (4) (5) (6) (7)

Application to Pinealocytes

Contain fluorogenic amines.

Pineal contains pools of 5HT and 5HTP [121] Exhibit amine precursor Pineal takes up TRP from uptake. circulation [204] Contain amino acid Pineal contains aromatic decarboxylase activity. L-amino acid decarboxylase [135] Such activity is present in Contain high levels of non-specific esterase aminergic sympathetic fibres in the pineal [10] activity. This is a characteristic Exhibit specific feature of pineal cells [272] immunofluoresence High aGPD activity is present Contain high levels of a-glycerophosphate re- in specific groups of ductase (aGPD) activity. pinealocytes [272] Masked metachromasia is Contain high present in pinealocytes [272] concentrations of sidechain carboxyl or carboxyamide groups (masked metachromasia).

Modified from [146].

termines when synthesis will occur, and its level, up to the saturation point of HIOMT [30,135]. The similar time courses of NAT activity and melatonin synthesis in the pineals of several species, and the similar characteristics of their light-induced suppression [40] lend further support to regulatory role for N A T in melatonin biosynthesis, as does the observation that HIOMT operates at only 80-90% of its in vitro capacity at night [119]. It may be that at certain times of the year, the adjustable production ceiling set by HIOMT will make it the pacemaker [30], but the same could be said of other factors whose availability varies with time, such as SAM, 5HT, and acetyl CoA (see, for example [253]). There appears little benefit in arguing for a single rate limiting enzyme, and it is clear that NAT controls access to melatonin biosynthesis. [48,157]. METABOLISMOF SEROTONIN:ALTERNATIVEPATHWAYS The alternative to N-acetylation for 5HT (Pathway I, Fig. 2) is oxidative deamination by MAO-A (Pathway II, Fig. 2) to the highly unstable 5-hydroxyindole acetylaldehyde, which is immediately either reduced to 5-hydroxytryptophol (5HTL) or oxidized to 5-hydroxyindole acetic acid (5HIAA). All four substances may be O-methylated by HIOMT (Fig. 2). The attention paid to melatonin has meant relative neglect for these alternative pathways; hence they are poorly understood, and their products d~sm~ssed as being of little biological importance, possibly because they do not match melatonin's influence on the reproductive system (see, for example [211]). The second pathway (Fig. 2), leading to acid metabolites, has been characterized as the major deactivation route for serotonin, but only recently has the third pathway, leading to the tryptophols, begun to receive attention. To dismiss this pathway and its products as nonfunctional in the absence of evidence is scientifically un-

sound, and may lead to serious omissions in our understanding of pineal function as a whole. It would seem judicious, therefore, to monitor closely the responses of the three pathways to a range of exogenous stimuli, and to observe the effects of these stimuli, and of the full range of pineal indole intermediates and products, on other endocrine tissues, and in biological fluids. This prompts the rhetorical question-why undertake such research? First hints that the pineal gland is involved in processes other than melatonin synthesis came from electrophysiological studies, which distinguished two types of pinealocytes; one type, whose electrical activity was inhibited by light (and which was involved in melatonin production), and another type, whose electrical activity was stimulated by light [235]. The same authors [234] have provided evidence obtained from extracellular recording electrodes for no less than five subtypes of pinealocytes, distinguished on the basis of their electrophysiological responsiveness (Table 2). A preliminary study, using intracellular recording from stimulated guinea pig pinealocytes [191] confirms the presence of two distinct cell types. In support of multiple pinealocyte cell types, some pinealocytes have been claimed to be centrally, rather than sympathetically innervated [74, 76, 173,219, 226] and there is evidence for at least some pinealocytes demonstrating circadian rhythmicity [220,232]. The release of more than one "extracellular message" was suggested by Klein et al. [136] in their description of "seesaw signal processing." This process involves "homologous sensitization" of cGMP response in the pinealocyte (i.e., maintenance of cGMP stimulation by continued neural input) suggested to be occurring simultaneously with homologous desensitization of a cAMP response (i.e., a depression of the response by continued neural stimulation). Such a seesaw regulation would allow modulation of a multicomponent response [136,300], and may be of great pineal significance in view of the suggested link [251] between cGMP and HIOMT, an enzyme involved in the formation of several putative pineal products. Seesaw signal processing must be distinguished from a suggested presynaptic action of cGMP to modulate NE release [195], a theory no longer popular [271]. Clearly, much remains to be learned of the role of cyclic nucleotides in mediating pineal endocrine responses [300]. Contributing to the neglect of pineal methylations other than those directly involved in melatonin formation was the observation that NAS is "the best substrate for H I O M T " in the original assay for HIOMT [9]. In this paper, eight possible alternative substrates were tested, of which only two (5HT and 5HIAA) were endogenous pineal, rather than synthetic, tryptamines. In subsequent literature, several potential substrates were dismissed [205] with little evidence of their suitability being examined [242]. Further, the applicability of HIOMT assays where the substrate NAS is supplied in excess must be questioned in light of the number of potential substrates in vivo. This issue becomes even more important if HIOMT does occur in the pineal in a number of separately regulated forms. This concept is quite plausible; we already know that several forms of NAT exist in the body, each with different regulatory mechanisms [13, 78, 115]. SIGNIFICANCEOF INDOLESOTHER THAN MELATONIN Once it is accepted that the pineal is capable of multicomponent responses, it becomes plausible to speak of simultaneous pineal involvement in both the classical

282

P I N E A L INDOLES PINEAL SYNTHETIC ACTIVITY Pinealocytes

Sympathetic nerve endings

APUD cellj

,f--~Oxidative deamination producing ( MAO-A Meiatonin; metabolism ) 5HT ~ producing (like EC-1 cells i .y-SHTL; of intestine)? / 5HIAA. "*" (presynaptic 5HT function ? )

FIG. 7. Proposed synthetic compartments in the mammalian pineal gland.

endocrine system and in the APUD (Amine precursor uptake and/or decarboxylation) system [88,193]. In this way, the pineal could act as a link between the neural and endocrine systems, facilitating the interaction of physiological processes and brain mechanisms with environmental input. Additionally, a local role for melatonin has been suggested [109]. Participation by the pineal gland in the APUD system would facilitate coordination of other, widely dispersed APUD components by the gland and thereby allow it to temper body homeostasis [48,149]. This view has been echoed (though without reference to APUD concepts) by other authors [19, 171,225]; the pineal has been suggested to play a major role in homeostatic regulation of brain monoamine levels [184], thermoregulation [125,208] and response to opioids [125-127]. Apart from possessing pools of 5HT and 5-hydroxytryptophan (5HTP), which suggested to Juillard and Collin [121] that the pinealocyte is involved in a "diffuse neuroendocrine system," the pinealocyte demonstrates all seven characteristics of the APUD cell (Table 3). APUD cells are found in such diverse tissues as the thyroid (C cells), pancreas (a, fl, ~ cells), pars distalis, and several cell types of the pituitary and the intestinal tract [146] APUD cells commonly produce both amines and peptides; for example the adrenal medulla releases epinephrine to influence cardiovascular and other physiological functions and simultaneously releases opioid/ACTH peptides directed at the central nervous system. It is tempting to hypothesize similar cooperative action for pineal indoles and peptides. The pineal represents an ideal model for studying such systems [52,55]; substances released from the pineal could act as third line effectors (after the somatic and autonomic nervous systems) with actions slower in onset and longer in duration than these two [193]. It has been suggested that N-acetylation (pathway I, Fig. 2) and oxidative deamination (pathways II and III) take place in different cells in the pineal [128]. The pinealocyte acts as a typical APUD cell, having an uptake mechanism for tryptophan and/or 5HTP, and synthesises 5HT. This product is then divided into two functional pools: that which is retained in the pinealocyte for NAS/melatonin synthesis, while the greater portion is returned to the synapse [276] and taken up by the sympathetic nerve endings for oxidative deamination. As various peptides have been demonstrated in the pinealocyte [104, 105, 178, 195, 199, 210, 214], it would appear that the APUD nature of the gland is localized within these cells;

whose amine products are 5HT and melatonin, as with the EC-1 APUD cells of the intestine [194]. In this model (Fig. 7), MAO-A-regulated metabolism is a discrete pineal process taking place in the sympathetic nerve-endings of the gland. The de-amination products of MAO-A include 5 H I A A - suggesting a presynaptic function for 5HT distinct from its role as the major indole precursor of the pineal--and 5HTL, which may be released into the synapse to further modulate pinealocyte activity [99]. Significantly, melatonin synthesis is elevated by inhibition of MAO type A, but not of type B [294]; the latter constitutes the larger portion of the MAO content of the pineal gland, and is localized almost entirely within the pinealocyte [187]. The above evidence increases the significance of pineal indole metabolism; instead of one single-purpose hormone (melatonin for reproduction) a variety of different responses, subserving different functions, is allowed [170, 196, 213]. There is increasing evidence for biological activity among nonmelatonin indoles.

N-Acetylserotonin (NAS) Probably possesses intrinsic activity, as well as being both a precursor and an important urinary metabolite [148,296] of melatonin. NAS has the ability to bind to both type-1 and type-2 5HT receptors in brain tissue [183], and demonstrates a different localization in the brain to melatonin [44,182]. Significantly, the diurnal rhythm in NAS concentration differs from the rhythm of melatonin in plasma [110,188], and a rise in NAT activity does not necessarily correlate with a rise in melatonin levels [130]. Seggie et al. [230] demonstrated that the responses of serum NAS and melatonin to different types of stress differ, and suggested that separate mechanisms exist to regulate the serum levels of these two indoleamines. Similarly, serum NAS and melatonin levels respond differently to the administration of the /3-adrenergic agonist isoproterenol [111]. NAS significantly depresses neuronal firing [236], and may in fact be responsible for some of the effects ascribed to melatonin [42, 181,296]. NAS has been shown to affect induced sleep patterns [249], to depress serum TSH levels [260], and to inhibit iodine accumulation by the thyroid gland [81]. For a review of the role of NAS in the central nervous system see [43].

5-Methoxytryptamine (5MT) Has some antigonadotrophic properties, is a potent agonist at 5HT receptors, and stimulates both aldosterone biosynthesis and cortisol release, suggesting the existence of a direct pineal-adrenal axis [179, 198, 242,272, 282].

5-Hydroxytryptophol (5HTL) 5HTL was first identified (in urine) in 1962 [142] and was suggested to mediate some of the sedative effects attributed to 5HT [257]. In the rat brain, 5HTL is localised almost entirely in the pineal gland [82], and in mice has been shown to induce sleep [92]. This latter observation is of interest in that one of the earliest manifestations of depressive illness in man is disturbance in normal sleep patterns (early morning waking), which could be associated with the ability of cyclic antidepressant drugs to actively suppress 5HTL synthesis (see, for example [185]). Fraschini et al. [100] demonstrated the existence in the median eminence of receptors specific for 5HTL, distinct

FOLEY, CAIRNCROSS AND FOLDES from 5HT receptors, which 5HTL is known to bind with high affinity [103]. These receptors were purported to play a role in luteinizing hormone secretion. In electrophysiological studies, 30% of non-Purkinje cerebellar cells were found to respond to microelectrophoretically-applied 5HTL [237]. Beck et al. [21-23] have examined another possible role for 5HTL in human behaviour. These workers have demonstrated in the clinic that plasma, urine and C.S.F. levels of 5HTL are elevated in chronic alcoholism and remain elevated for up to 21 days following withdrawal. However, in social drinking, whilst 5HTL levels are similarly elevated, such an elevation is not maintained for more than 24 hours. These latter observations, when viewed in conjunction with findings of Semm and Vollrath [236,237] who showed that 5HTL depressed pineal electrical activity, suggest a role for 5HTL in the regulation of pinealocyte activity. However, further work is required before definitive conclusions can be made. 5HTL is synthesised in significant amounts in the liver, lung and intestines of the rat [64], and outside the pineal gland can exist in a conjugate form, from which it may be liberated by treatment with a sulphatase with /3-glucouronidase activity. Thus, about 13% of cerebrospinal 5HTL is conjugated [21] and almost all plasma and urinary 5HTL exists in this form [22,24]. Pineal 5HTL appears not to exist in the conjugate form [24], and very recent observations have indicated a circadian rhythm for pineal 5HTL, the indole concentration increases sixfold during the middle of the light period and declining dramatically five hours prior to the onset of darkness [99].

5-Methoxytryptophol (5MTL) Exhibits antigonadotrophic properties in rats during estrus and puberty, and also under conditions of stress and insulin-induced hypoglycemia. 5MTL appears to be active at circulating levels only ten percent of those melatonin [59]. In contrast to melatonin, 5MTL exhibits a concentration maximum during the light-period, possibly due to rapid synthesis and release [290]. Pineal and plasma levels of 5MTL do not always correlate (both are melatonin-independent), but peripheral deacetylation of O-acetyl 5-methoxytryptophol (aMTL) or conversion of another indole, may obscure any rhythm [10,156]. Like melatonin, 5MTL stimulates cortical electrical activity [236], though acting on different cells, and depresses that of cerebellar cells [237]. In some cases, 5MTL appears to exhibit greater antigonadotrophic activity than melatonin [216].

O-Acetyl 5-Methoxytryptophol (aMTL) Blocks both types of cholinergic receptor, by an effect on membrane activity rather than direct antagonism [242]; the concentration of aMTL required for this effect necessitates that the target cells be located in the near proximity of the pineal. The aMTL pathway is possibly analogous to the melatonin route [15] but remains little investigated, perhaps due to the lack of commercial availability of O-acetyl derivatives at the present time [10, 15, 16, 244]. Thus the relatively few reports of indole bioactivity have shown positive effects for indoles other than melatonin in both endocrine and APUD function (see [239], and, for modification of the gateway enzyme MAO-A, see [25,263]). Pineal indoleamines may also act as releasing factors for peptides [109]; most methoxyindoles appear to enhance formation of peptide-containing granular vesicles. Outside the scope of this review, but vital to an overview of pineal func-

283 TABLE 4 ADVANTAGESOF HPLC/ELECTROCHEMICALDETECTION (HPLC/ED) AS A SEPARATION/MEASUREMENTSYSTEMFOR INDOLES (1) Capacity for high resolution by HPLC in reverse phase mode. (2) High sensitivity of electrochemical detection. (3) Minimal sample preparation required, reducing the time available for chemical modification or degradation. (4) Rapidity of measurement. (5) Over a dozen different pineal compounds assayable in only three assays. Modified from [146].

tion as a whole, is the identification in the pineal gland of a range of peptides including oxytocin (OT), arginine vasopressin (AVP), and, controversially, arginine vasotocin (AVT); the significance of these peptides (and numerous other peptides and releasing factors) in the pineal is not yet understood [104, 105, 180, 197, 202, 203]. To date, at least twelve, and perhaps more than twenty indoles of pineal origin have been identified or suggested, most present at about 100 pg/mL in plasma [213]. D E V E L O P M E N T OF M E A S U R E M E N T T E C H N I Q U E S FOR I N D O L E A M I N E S AND T H E I R SYNTHETIC ENZYMES With our increasing awareness of the sensitivity of the pineal gland to a wide variety of stress and external stimuli (see, for example [47]) the need for extreme care and consistency in all aspects of pineal study is becoming increasingly evident. The most widely used NAT assay [80] has been proved reliable, and acceptable for most applications; however, the observation that 5MT is a better substrate for NAT than is 5HT, cast doubts on the assumed melatonin pathway [78], Klein and coworkers developed a two-dimensional thin layer chromatography (TLC) assay for rat pineal NAT activity which yields good separation of a range of isotopically labelled methoxyindoles [137]. This technique has also been successfully used in studies of ovine pineal function (A. Foldes, unpublished observations). Rollag [223] presented a state of the art survey of indole measurement techniques, consisting mostly of radioimmunoassays (RIA) for melatonin and NAS. The priorities of the time are indicated by the absence of assays for other indoles. This followed Ebels' earlier "chemical study of some biologically active pineal fractions" [86] which presented a useful separation of pineal components, but made little attempt to identify or quantitate the separate fractions. A specific RIA for NAS had been developed by 1981 [189]. In the meanwhile, however, the accuracy and/or specificity of RIAs for melatonin had been called into question in 1980 when twelve laboratories, independently assaying samples from a common pool of human serum, produced divergent results [284]. A more robust assay designed for both clinical and experimental purposes has recently been reported [278]. The traditional HIOMT assay suffers from its assumption of a single major endogenous substrate; and, as Quay [205] noted, from a lack of appreciation of certain technical factors

284

FOLEY. CAIRNCROSS AND FOLDES TABLE 5 DETECTION LIMITS(IN PICOGRAMS}OF METHOXY-AND HYDROXYINDOLESWITH HPLC SEPARATION

Indole

Amperometric Detection

Fluorimetric Detection

Normal Pineal Ranges (pg/pineal)

15~ 5~ 5~ l0 t 101 20~ 20 ~ 20~ -10~ 201

202 -72 35~ 203 403 252 -303 203 --

4,000-12,000 ? 30,000-50,000 2,000-18,000 0- 4,000 0- 6,000 0- 3,000 ? 0- 100 0- 100 ?

Tryptophan 5-Hydroxytryptophan 5-Hydroxytryptamine 5-Hydroxyindoleacetic Acid 5-Hydroxytryptophol N-Acetyl Serotonin Melatonin 5-Methoxytryptophan -Methoxyindole Acetic Acid 5-Methoxytryptophol 6-Hydroxymelatonin

References: 1 = Mefford and Barchas [168]; 2 = Anderson et al. [4]; 3 = Anderson et al. [6]. Normal ranges are estimated from the levels found by the papers cited.

in the preparation of the assay and the provision of substrate and/or methyl donor at sub-saturation levels. To take into account the possibility of multiple HIOMT isozymes and to ensure the presence of all endogenous substrates in physiological concentrations, Balemans et al. [16] published a new method for measuring the methylating capacity of the pineal gland. The homogenized gland is incubated with radioactively labelled SAM, but no exogenous substrate is added. Following methylation of the endogenous substrates, the labelled methoxyindoles are separated by TLC. This method has been used to study the effects of pteridines and of different wavelengths of light on HIOMT activity [11-14, 17, 18, 199]. The TLC separation is adequate for most methoxyindoles, but fails to separate melatonin from MTL. A two-dimensional TLC technique which resembles that used by Klein et al. [137] for assaying NAT activity (see above) and which also separates hydroxyindoles, has supported the coexistence of HIOMT isozymes [175]. The same authors [175] have further suggested that melatonin production by rat pineal gland cultures may be regulated by availability of the precursor NAS, whereas production of other methoxyindoles appeared to be precursor independent. Future advances, especially in high performance TLC [29], may lead to wider applications of this technique [68]. The most promising analytical technique for providing a rapid, precise measurement of endogenous indoles appears to be high performance liquid chromatography (HPLC), coupled with electrochemical detection (ED; see Table 4). Hydroxyindole analysis in urine using reverse phase (RP) HPLC was first described by Graffeo and Karger [106] in 1976 using fluorescence detection. The technique was extended by Anderson and others [3,5] to measure tryptophan (TRP) metabolites in urine, CSF, plasma and brain, with particular reference to TRP metabolism in the mentally disturbed; for this reason, the range of pineal-related indole compounds assayed was limited to TRP, 5HT, 5HIAA and (later) melatonin. Advances in amperometric detection advanced the ap-

propriateness of electrochemical detection in the assay of aromatic compounds by RP-HPLC [131,132]. In subsequent studies, Anderson et al. [4,5] used fluorometric and amperometric detectors in series to study TRP metabolism in rat brain, and now in the pineal specifically, but despite obtaining better detection limits for some indoles by amperometry (Table 5), they returned to using fluorometry alone [6,295]. Using fluorometric detection, Anderson and co-workers described a photoperiod-dependent rhythm in six pineal indoles, and showed that only one half of the SCG was required to maintain this rhythm [267,295]. Krstulovic et al. [141] also used HPLC coupled with both detection systems, because ultraviolet/fluorescence detection was adequate to quantitate the kynurenine metabolites of TRP, but was not sufficiently sensitive to monitor other serum indoles. They also showed that deproteinization is the only preparation necessary before injection of serum onto the HPLC column. Several Japanese laboratories published enrichment methods for the assay of serum hydroxyindoles [ll3, 114, 174, 181] and a method using ion-pair HPLC with fluorometric detection was reported [l], but most authors have found that ED or amperometric detection provides a more sensitive analysis and greater versatility of application. The technique (HPLC-ED) has now been widely applied to the determination of many different biomolecules, including indoles, catecholamines, enzyme activities, pharmacological agents, hormones and even peptides ([94,153]; for reviews see [248,270]). Diversity of application and technical improvements in commercially available units, leading to increased sensitivity render the technique a viable, attractive proposition for measurement of a range of endogenous and exogenous substances at low concentrations (picomolar) in a number of fields, including the study of pineal function. Mefford and Barchas [168] were among the first to couple HPLC (RP) with ED to measure six hydroxy- and five methoxyindoles in brain and pineal tissue at detection limits of 5-25 pg, levels well below tissue concentrations for most indole compounds (see Table 5). This is the most sensitive

PINEAL INDOLES

285

and complete indole analysis published to date, demonstrating the superiority of the method over fluorescence detection, gas chromotography-mass spectrometry and RIA. Their method was also applicable to other biological fluids (urine, plasma or saliva), protein precipitation again being the only required sample preparation. With a slightly modified technique applicable to single or pooled pineal glands [169], they determined the circadian rhythms of TRP, 5HT, 5HTL, 5HIAA, NAS, 5MTL, 5MIAA and melatonin, with applicability also to 5HT, aMTL, 5MT and 5MTP. Their results showed basic agreement with, as well as some slight differences from, the results of Young and Anderson [295] obtained using fluorometric detection. Caliguri and Mefford [49] reported a microbore HPLC technique allowing quantification of 50--200 fg of 5HT, 5HIAA, 5HTP, NE, E, DA and 3,4-dihydroxyphenylacetic acid (DOPAC). A similar method has been used to examine the effects of various drugs on 5HT metabolism and on 5HT present during the light period in the granular vesicles of pinealocytes [129]. The conventional HPLC method has been further elaborated by Champney and co-workers [60,247] who performed the following assays on a single pineal gland: N A T assay (modified Deguchi and Axelrod [80], 1/10 gland), HIOMT assay (modified Axelrod and Weissbach [9], 1/10 gland), RIA for melatonin (Rollag and Niswender [224], 1/2 gland) and HPLC for hydroxyindoles (Mefford and Barchas [168], 3/10 gland). In this way much more information is extracted from a single gland than has hitherto been thought possible, although methoxyindoles other than melatonin have not been investigated. The surprising find from this close examination has been that correlation between the various pineal components in individual animals has not been very high, suggesting that pineal synthesis of the various indoles measured is not continuous, but responsive to pulsatile N E release from the sympathetic nerves in line with that already demonstrated in man [268,279]. More than ever before, the technology is now available to investigate the endocrine function of the pineal gland and to develop a complete profile of its indole constituents. In addition to better equipment with increased sensitivity, a number of methodological improvements have also assisted the progress of this work. These include the use of ascorbic acid and of EDTA in all solvents to prevent auto-oxidation of indoles and to extend their storage life [269]; determination of optimal isocratic conditions for detection of hy-

droxyindoles, with respect to pH, elution rate, solvent and buffer concentrations, oxidation potential and so on [91]; and the use of red light when excising pineal glands to minimize changes in indole concentration and enzyme activities [102]. SUMMARY When " N e w Horizons in Pineal Research" [215] appeared nine years ago, there were intimations that melatonin may prove to be only one o f several active pineal indole constitutents, each having different physiological roles; the techniques for examining such a proposition were, however, not yet fully assembled. The pineal gland is now known to be associated with a wide range of physiological phenomena, and to be a complex, highly specialized organ of multiple efficacy. The various indoles synthesized by the pineal may be implicated in a system whereby the pineal gland, and hence the brain, perceives, differentiates and integrates environmental information. Pevet [198] lists three possible modes of action for pineal indoles: (1) as transmitter substances modifying their cells of origin, (2) as modulators of neighbouring cells, (3) as hormones modifying distant target organs reached via the general circulation. Such roles have already been established for two indoles, melatonin and 5MT. Further physiological functions for pineal indoles may involve long term regulation and cyclic changes of subtle activity whose measurement is less amenable to detection and detailed investigation. However, a better understanding of the role of the pineal gland is now accessible through our increasing knowledge of its biochemistry and through our possession of the tools with which to investigate its responses to a variety of environmental stimuli by accurate measurement of its indole products in the pineal itself, in CSF and in the circulation. The pineal body, with its central role in neural and in endocrine function, its diffuse realms of influence, and its unique role in the conversion of environmental stimuli into physiological responses, is no longer a black box we cannot explore; nor is it a trivial curiosity we can afford to ignore. ACKNOWLEDGEMENTS The authors gratefully acknowledge the contributions of Professor Emeritus Charles Vernon and Dr. B. E. C. Banks for their constructive criticism of the manuscript. The authors also thank Mrs. S. Hummelstad for the typing of this manuscript.

REFERENCES

I. Adell, A., J. M. Tusell, F. Artigas, E. Martinez, G. Sunol and E. Gelpi. Fluorometric determination of tryptophan and its brain indoleamine metabolites by ion-pair high performance liquid chromatography. J Liquid Chromatogr 6: 527-541, 1983. 2. Alphs, L., A. Weller and W. Lovenberg. Adrenergic regulation of the reduction in acetyl co-enzyme A: arylamine N-acetyltransferase activity in the rat pineal. J Neurochem 34: 83-90, 1980. 3. Anderson, G. M. and W. C, Purdy. Liquid chromatographyHuorometric system for the determination of indoles in physiological samples. Anal Chem 51: 283-286, 1979. 4. Anderson, G. M., J. G. Young, D. K. Batter, S. N. Young, D. J. Cohen and B. A. Shaywitz. Determination of indoles and catechols in rat brain and pineal using liquid chromatography with fluorometric and amperometric detection. J Chromatogr 223: 315-320, 1981.

5. Anderson, G. M., J. G. Young and D. J. Cohen. Rapid liquid chromatographic determination of tryptophan, tyrosine, 5-hydroxyindoleacetic acid and homovanillic acid in cerebrospinal fluid. J Chromatogr 164: 501-505, 1979. 6. Anderson, G. M., J. G. Young, D. J. Cohen and S. N. Young. Determination of indoles in human and rat pineal. J Chromatogr 228: 155-163, 1982. 7. Anton-Tay, F. Melatonin effects on brain function. Adv Biochern Psychopharmaco[ l l : 315-324, 1974. 8. Arendt, J., S. Hampton, J. English, P. Kwasowski and V. Marks. 24-Hour profiles of melatonin, cortisol, insulin, C-peptide and GIP following a meal and subsequent fasting. Clin Endocrinol (Oxf) 16: 8%95, 1982. 9. Axelrod, J. and M. Weissbach. Purification and properties of hydroxyindole-O-methyl transferase. J Biol Chem 236: 211213, 1961.

286 10. Balemans, M. G. M. Indole metabolism in the pineal gland, the Harderian gland and the retina of mammals. In: The Pineal Organ: Photobiology, Biochronornetry, Endocrinology, edited by A. Oksche and P. Pevet. Amsterdam: Elsevier, North Holland Biomedical Press, 1981, pp. 261-280. 11. Balemans, M. G. M., I. Ebels, H. G. Hendriks and M. F. Van Berlo. Changes in the circadian rhythmicity of hydroxyindoles and methoxyindoles in the pineal gland of 28-day old Wistar rats exposed to white, red and green light. J Neural Transm 53: 293-303, 1982. 12. Balemans, M. G. M., I. Ebels, H. G. Hendriks, M. van Berlo and A. de Moree. Influence of some pteridines on pineal 5-methoxyindole synthesis in male Wistar rats periodically exposed to either white or red light. J Neural Transm 56: 199-210, 1983. 13. Balemans, M. G. M., I. Ebels, H. G. Hendriks, M. van Berlo and A. de Moree. The influence of some pteridines on pineal 5-methoxyindole synthesis in male Wistar rats periodically exposed to either white or green light. J Neural Transm 58: 121134, 1983. 14. Balemans, M. G. M., W. C. Legerstee and J. van Benthem. Day and night rhythms in the methylation of N-acetyiserotonin/5 hydroxytryptophol in the pineal gland of male rats of different ages. J Neural Transm 45: 265-272. 1979. 15. Balemans, M. G. M., D. Mans, I. Smith and J. Van Benthem. The influence of GABA on the synthesis of N-acetylserotonin, melatonin, O-acetyl-5-hydroxytryptophot and O-acetyl-5methoxytryptophol in the pineal gland of the male Wistar rat. Reprod Nutr Dev 23: 151-160, 1983. 16. Balemans, M. G. M., E. M. Noordegraff, F. A. M. Bary and M. F. van Berlo. Estimation of the methylating capacity of the pineal gland with special reference to indole metabolism. Experientia 34: 887-888, 1978. 17. Balemans, M. G. M., P. Pevet, J. van Benthem, C. HalderMisra, I. Smith and H. Hendriks. Day/night rhythmicity in the methylating capacities for different 5-hydroxyindoles in the pineal, the retina and the harderian gland of the golden hamster (Mesocricetus auratus) during the annual seasons. J Neural Transm 56: 53-72, 1983. 18. Balemans, M. G. M., J. van Benthem, W. C. Legerstee, A. de Moree, H. J. P. M. Noteborn and I. Ebels. The influence of some pterins on the circadian rhythmicity of hydroxindole-Omethyl transferase in the pineal gland of 42-day old male Wistar rats. Reprod Nutr Dev 20: 1051-1060, 1980. 19. Bartness, T. J. and G. N. Wade. Photoperiodic control of bodyweight and energy metabolism in Syrian hamsters (Mesocricetus auratus): role of pineal gland, melatonin, gonads and diet. Endocrinology 114: 492-498, 1984. 20. Bartsch, C., H. Bartsch, A. J. Jain, K. R. Laumas and L. Wetterberg. Urinary melatonin levels in human breast cancer patients. J Neural Transm 52: 281, 1981. 21. Beck, O., S. Borg, G. Edman, B. Fyrr, G. Oxenstierna and G. Sedvall. 5-Hydroxytryptophol in human cerebrospinal fluid: conjugation, concentration gradient, relationship to 5-hydroxyindoleacetic acid, and influence of hereditary factors. J Neurochem 43: 58--61, 1984. 22. Beck, O., S. Borg, L. Eriksson and A. Lundman. 5-Hydroxytryptophol in the cerebrospinal fluid and urine of alcoholics and healthy subjects. Naunyn Schmiedebergs Arch Pharmacol 321: 293-297, 1982. 23. Beck, O., S. Borg, B. Holmstedt and H. Stibler. Levels of 5-hydroxytryptophol in cerebrospinal fluid for alcoholics determined by gas chromatography-mass spectrometry. Biochem Pharmacol 29: 693-696, 1980. 24. Beck, O., S. Borg, G. Jonsson, A Lundman and P. Valverius. Measurement of 5-hydroxytryptophol and 5-hydroxyindoleacetic acid in human and rat brain and plasma. J Neural Transm 59: 57-67, 1984. 25. Beck, O., G. Jonsson and A. Lundman. 5-Methoxyindoles in pineal gland of cow, sheep, pig and rat. Naunyn Schmiedbergs Arch Pharrnacol 318: 49-55, 1981.

FOLEY, CAIRNCROSS AND FOLDES 26. Belokrylov, G. A., V. G. Morozov, V. Khavinson and B. N. Sofronov. Effect of low-molecular-weight extracts of heterologous thymus and pineal glands and hypothalamus on the immune response in mice. Byul Eksp Biol Med 81: 202-204, 1978.

27. Berg, G. T. and D. C. Klein. Norepinephrine increases the [32P]-labelling of a specific phospholipid fraction of postsynaptic pineal membranes. J Neurochem 19: 2519-2532, 1972. 28. Berridge, M. J. Inositol triphosphate and diacylglycerol as second messengers. Biochem J 220: 345--360, 1984. 29. Bertsch, W., S. Hara, R. E. Kaiser and Z. Zlatkis (Ed). Instrumental HPTLC. Heidelberg: Dr. Alfred Hfithig Verlag, 1986. 30. Binkley, S. Pineal biochemistry--comparative aspects and circadian rhythms. In: The Pineal Gland, Vol 1: Anatomy and Biochemistry, edited by R. J. Reiter. Boca Raton, FL: CRC Press Inc., 1981, pp. 155-172. 31. Binkley, S. Circadian rhythm in pineal NAT activity: phase shifting by light pulses (II). J Neurochem 41: 273-276, 1983. 32. Binkley, S. Circadian rhythms of pineal function in rats. Endocr Rev 4: 225-270, 1983. 33. Binkley, S., D. C. Klein and J. L. Weller. Pineal SNAT activity: protection of stimulated activity of acetyl-CoA and related substances. J Neurochem 26: 51-55, 1976. 34. Birau, N. Melatonin rhythms in human serum: progress in screening, investigation and clinic. In: Melatonin----Current Status and Perspectives, edited by N. Birau and W. Schlot. Oxford: Pergamon Press, 1981, pp. 297-326. 35. Birau, N. Advances in human melatonin research-physiological and clinical aspects. IRCS Med Sci Biochem 13: 1-5, 1985. 36. Birkland, A. J. Plasma melatonin levels and nocturnal transitions between sleep and wakefulness. Neuroendocrinology 34: 126--131, 1982. 37. Bittman, E. L., R. J. Dempsey and F. J. Karsch. Pineal melatonin secretion drives the reproductive response to day length in the ewe. Endocrinology 113: 2276-2283, 1983. 38. Bittman, E. L., F. J. Karsch and F. W. Hopkins. Role of the pineal gland in ovine photoperiodism: regulation of seasonal breeding and negative feedback effects of estradiol upon luteinizing hormone secretion. Endocrinology 113: 329-336, 1983. 39. Blumberg, J. B., R. E. Taylor and F. Sulser. Blockade by pimazide of NA-sensitive adenylate cyclase in the limbic forebrain. J Pharm Pharmacol 27: 125-128, 1975. 40. Brainard, G. C., B. A. Richardson, T. S. King, S. A. Matthews and R. J. Reiter. The suppression of pineal melatonin content and NAT activity by different light irradiances in the Syrian hamster: a dose-response relationship. Endocrinology 113: 293-296, 1983. 41. Brainard, G. C., B. A. Richardson, T. S. King and R. J. Reiter. The influence of different light spectra on the suppression of pineal melatonin in the Syrian hamster. Brain Res 294: 333339, 1984. 42. Brown, G. M., J. Basinska, J. Bubenik, D. Sibany, L. J. Grota and H. C. Stancer. Gonadal effects of pinealectomy and immunization against N-acetylindolealkylamines in the hamster. Neuroendocrinology 22: 289-297, 1976. 43. Brown, G. M., O. Pulido, L. J. Grota and L. P. Niles. N-acetylserotonin in the central nervous system. Prog Neuropsychopharmacol Biol Psychiatry 8: 475-480, 1984. 44. Brown, G. M., O. Pulido, L. P. Niles, S. Psarakis, A. Parietis, G. A. Bubenik and L. J. Grota. Differential localization of melatonin and N-acetylserotonin in brain. In: The Pineal Gland and its Endocrine Role, edited by J. Axelrod, F. Fraschini and G. P. Velo. New York: Plenum Press, 1983, pp. 257-275. 45. Buda, M. and D. C. Klein. A suspension culture of pinealocytes--regulation of NAT activity. Endocrinology 103: 1483-1493, 1978. 46. Buswell, R. W. The pineal and neoplasia. Lancet 1: 34, 1975.

PINEAL INDOLES 47. Cairncross, K. D. Olfactory bulbectomy as a model of ,epression. In: Animal Models in Psychopathology, edited by N. Bond. Sydney: Academic Press, 1984, pp. 99-128. 48. Cairncross, K. D. and P. Foley. Does the pattern of 5-hydroxytryptophol (5HTL) synthesis in the rat pineal gland indicate an APUD function for the gland? Proc Aust Soc Med Res 18: 31, 1985. 49. Caliguri, E. J. and I. N. Mefford. Femtogram detection limits for biogenic amines using microbore HPLC with electrochemical detection. Brain Res 296: 156-159, 1984. 50. Calvo, J. and J. Boya. Ultrastructure of the rat pineal stalk. Acta Anat (Basel) 123: 172-177, 1985. 51. Cardinali, D. P. Hormone effects on pineal gland. In: The Pineal Gland, Vol 1: Anatomy and Biochemistry, edited by R. J. Reiter. Boca Raton, FL: CRC Press Inc., 1981, pp. 243-272. 52. Cardinali, D. P. Molecular mechanisms of neuroendocrine integration in the central nervous system: an approach through the study of the pineal gland and its innervating sympathetic pathway. Psychoneuroendocrinology 8: 3-30, 1983. 53. Cardinali, D. P. and M. N. Ritta. The role of prostaglandins in neuroendocrine junctions. Studies in the pineal gland and the hypothalamus. Neuroendocrinology 36: 152-160, 1983. 54. Cardinali, D. P., M. N. Ritta, E. Pereyra and C. G. Solveyra. Role of prostaglandins in rat pineal neuroeffector junction: changes in melatonin and norepinephrine release in vitro. Endocrinology 111: 530-534, 1982. 55. Cardinali, D. P., M. N. Ritta, M. I. Vacas, P. R. Lowenstein, P. V. Gejman, C. G. Solveyra and E. Pereyra. Molecular aspects of neuroendocrine integrative processes in the pineal gland. In: The Pineal Gland and its Endocrine Role, edited by J. Axelrod, F. Fraschini and G. P. Velo. New York: Plenum Press, 1983, pp. 199-215. 56. Cardinali, D. P. and M. I. Vacas. Feedback control of pineal function by reproductive hormones---a neuroendocrine paradigm. J Neural Transm [Suppl] 13" 175-201, 1978. 57. Carmen, J. S., R. M. Post, R. Buswell and F. K. Goodwin. Negative effects of melatonin on depression. A m J Psychiatry 133: 1181-1186, 1976. 58. Carter, D. S., V. D. Hall, L. Tamarkin and B. D. Goldman. Pineal is required for testicular maintenance in the Turkish hamster (Mesocricetus brandti). Endocrinology l l h 863-871, 1982. 59. Carter, S. J., C. A. Laud, I. Smith, R. M. Leone, R. E. Silman, R. J. L. Hooper, D. L. Larson-Carter, M. D. A. Finnie and P. E. Mullen. 5-methoxytryptophol in rat pineal glands and other tissues. Prog Brain Res 52: 267-269, 1979. 60. Champney, T. H., A. P. Holtorf, R. W. Steger and R. J. Reiter. Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland. J Neurosci Res 11: 59-66, 1984. 61. Chan, A. and M. Ebadi. The kinetics of NE-induced stimulation of SNAT in bovine pineal gland. Neuroendocrinology 31: 244--251, 1980. 62. Chan, A. and M. Ebadi. Evidence for existence of a SNAT inactivating substance in rat pineal gland. Endocr Res Commun 8: 205-227, 1981. 63. Charbannes, B., N. Sarda, L. Gronenberger and H. Pacheco. Diurnal variations of S-adenosyl-L-methionine and adenosine content in rat pineal gland. Life Sci 35: 589-596, 1984. 64. Cheifetz, S. and J. J. Warsh. Occurrence and distribution of 5-hydroxytryptophol in the rat. J Neurochem 34: 1093-1099, 1980. 65. Cohen, M., M. Lippman and B. Chabner. Role of pineal gland in aetiology and treatment of breast cancer. Lancet ii: 814--816, 1978. 66. Corderoy-Buck, S. Pineal HIOMT regulation in the albino rat. Unpublished honors thesis, Macquarie University, Sydney, Australia, 1983. 67. Corderoy-Buck, S. and K. D. Cairncross. Involvement of the beta one adrenoceptor in pineal hydroxindole-O-methyl transferase activation following acoustic alarm in the rat. Neuroendocrinol Lett 6: 129-133, 1984.

287

68. Craft, C. M. and R. J. Relter. Derivatives of [3H]serotonin in organ cultures of hamster pineal glands. Life Sci 34: 1775-1782, 1984. 69. Cramer, H., J. Rudolph, U. Consbruch and K. Kendel. On the effect of melatonin on sleep and behaviour in man. Adv Biochem Psychopharmacol 11: 187-191, 1974. 70. Cremer-Bartels, G. The effect of pteridines on multiple forms of HIOMT in the retina and the pineal gland. Prog Brain Res 52: 231-239, 1979. 71. Cremer-Bartels, G. and I. Ebels. Pteridines as nonretinal regulators of light-dependent melatonin biosynthesis. Proc Natl Acad Sci USA 77: 2415-2418, 1980. 72. Cunnane, S. E., M. S. Manku and D. F. Horrobin. The pineal and regulation of fibrosis: pinealectomy as a model of primary biliary cirrhosis: roles of melatonin and prostaglandins in fibrosis and regulation of T lymphocytes. Med Hypotheses 5: 403-414, 1979. 73. Dafny, N. Electrophysiological evidence of photic, acoustic and central input to the pineal body and hypothalamus. Exp Neurol 55: 449-457, 1977. 74. Dafny, N. Photic input to rat pineal gland conveyed by both sympathetic and central afferents. J Neural Transm 48: 203208, 1980. 75. Dafny, N. Two photic pathways contribute to pineal evoked responses. Life Sci 26: 737-742, 1980. 76. Dafny, N. Evidence that the rat pineal has neuronal connections via the pineal stalk. Exp Neurol 70: 858--861, 1983. 77. Dafny, N., R. McClung and S. J. Strada. Neurophysiological properties of the pineal body. I. Field potentials. Life Sci 16: 611-620, 1975. 78. Deguchi, T. Characteristics of serotonin-acetyl co-enzyme A-N-acetyltransferase in pineal gland of rat. J Neurochem 24: 1053-1056, 1975. 79. Deguchi, T. Sympathetic regulation of circadian rhythm of SNAT activity in pineal gland of infant rat. J Neurochem 38: 797-802, 1982. 80. Deguchi, T. and J. Axelrod. Sensitive assay for serotonin N-acetyltransferase activity in rat pineal. Anal Biochem 50: 174--179, 1972. 81. De Prospo, N. D., R. J. Safinski, L. J. de Martino and E. T. McGuinnes. Melatonin and its precursor's effect on ~alI uptake by the thyroid gland under different photic conditions. Life Sci 8: 837-842, 1969. 82. Diggory, G. L., P. M. Ceasar, D. Hazelby and K. T. Taylor. Endogenous 5-hydroxytryptophol in mouse brain. J Neurochem 32: 1323-1325, 1979. 83. Dilman, V. M. Metabolic immunodepression and carcinogenesis. Mech Ageing Dev 8: 153-173, 1978. 84. Dilman, V. M., V. N. Anisimov, M. N. Ostroumova, V. K. H. Khavinson and V. G. Morozov. Increase in lifespan of rats following polypeptide pineal extract treatment. Exp Pathol 17: 539-545, 1979. 85. Ebadi, M., A. Chan, H. Hammad, P. Govitrapong and S. Swanson. SNAT and its regulation by pineal substances. Prog Clin Biol Res 92: 233-241, 1982. 86. Ebels, I. A chemical study of some biologically active pineal fractions. Prog Brain Res 52: 309-321, 1979. 87. Ehrenkranz, J. R. L., L. Tamarkin, F. Comite, R. E. Johnsonbough, D. E. Bybee, D. L. Lorioux and G. B. Cutler, Jr. Daily rhythm of plasma melatonin in normal and precocious puberty. J Clin Endocrinol Metab 55: 307-310, 1982. 88. Epple, A. Functional principles of vertebrate endocrine systems. Verh Dtsch Zool Ges 73: 117-126, 1982. 89. Erlich, S. S. and M. L. J. Apuzzo. The pineal gland--anatomy, physiology and clinical significance. Review. J Neurosurg 63: 321-341, 1985. 90. Fahrenkrug, J., U. Haglund, M. Jodal, O. Lundgren, L. Olbe and O. B. Schaffalitzky de Muckadell. Nervous release of vasoactive intestinal polypeptide in the gastrointestinal tract of cats: possible physiological implications. J Physiol (Lond) 284: 291-295, 1978.

288 91. Falkowski, A. J. and R. Wei. Optimized isocratic conditions for the simultaneous determination of serotonin precursors and metabolites by reversed-phase-high pressure liquid chromatography with electrochemical detection. Anal Biochem 115: 311-317, 1981. 92. Feldstein, A., F. H. Chang and J. M. Kucharski. Tryptophol, 5-hydroxytryptophol and 5-methoxytryptophol induced sleep in mice. Life Sci 9: 323-329, 1970. 93. Fevre-Montange, M., E. van Cauter, S. Refetoff, D. Desir, J. Tourniaire and G. Copinschi. Effects o f " j e t lag" on hormone patterns. II. Adaptation of melatonin circadian periodicity. J Clin Endocrinol Metab 52: 642-649, 1981. 94. Fisher, L. A. and J. D. Fernstrom. Measurements of nonapeptides in pineal and pituitary using reversed-phase, ion-pair liquid chromatography with post-column detection by radioimmunoassay. Life Sci 28: 1471-1481, 1981. 95. Fiske, V. M., K. L. Parker, R. A. Ulmer, C. H. Ow and N. Aziz. Effect of melatonin alone or in combination with human chorionic gonadotropin or ovine luteinizing hormone on the in vitro secretion of estrogens or progesterone by granulosa cells of rats. Endocrinology 114: 407-410, 1984. 96. Foldes, A., R. M. Hoskinson, R. J. Scaramuzzi, N. T. Hinks and C. A. Maxwell. Modification of sheep pineal fl-adrenoceptors by some gonadal steroids but not by melatonin. Neuroendocrinology 37: 378--385, 1983. 97. Foldes, A., C. A. Maxwell, A. J. Rintoul and R. W. Edols. Sheep pineal beta-adrenoceptor function--interaction with gamma-aminobutyric acid. Neuroendocrinology 38: 206-211, 1984. 98. Foldes, A., C. A. Maxwell, R. J. Scaramuzzi, J. B. Donnelly, R. M. Hoskinson and A. J. Rintoul. Seasonal changes in the sensitivity of ovine pineal/3-adrenoceptors to steroids. Neuroendocrinology, in press, 1985. 99. Foley, P. and K. D. Cairncross. Circadian synthesis of 5-hydroxytryptophol in the pineal gland of the rat. In: Current Status and Perspectives in Pineal Research EPSG Symposium, 1985, p. 33. 100. Fraschini, F., B. Mess, F. Piva and L. Martini. Brain receptors sensitive to indole compounds: function in control of luteinizing hormone secretion. Science 159:1104-1105, 1968. 101. Friedman, E., F. D. Yocca and T. B. Cooper. Antidepressant drugs with varying pharmacological profiles alter rat pineal beta-adrenergic-mediated function. J Pharmacol Exp Ther 228: 545-550, 1984. 102. Frowein, A. and V. Lapin. Effects of sham-pinealectomy, performed under white and red lights, on the melatonin content of rat pineal glands. Experientia 35: 1681, 1979. 103. Fu, L. H. W., S. Hayashi and N. Toda. Effects of 5-hydroxytryptophol, a 5-hydroxytryptamine metabolite, on isolated cerebral arteries of the dog. Br J Pharmacol 69: 17-18, 1980. 104. Gauqnelin, G., G. Geelen, F. Louis, A. M. Allevard, C. Meunier, G. Cuisinaud, S. Benjanet, N. G. Seidah, M. Chretien, J. J. Legros and C. Aharib. Presence of vasopressin, oxytocin and neurophysin in the retina of mammals, effect of light and darkness, comparison with the neuropeptide content of the neurohypophysis and the pineal gland. Peptides 4: 509515, 1983. 105. Geelen, G., A. M. Allevard Burguburu, G. Gauquelin, Y. Z. Xiao, J. Frutoso, C. L. Qharig, B. Sempore, C. Meunier and G. Augoyard. Radioimmunoassay of arginine vasopressin, oxytocin and arginine vasotocin-like material in the human pineal gland. Peptides 2: 45%466, 1981. 106. Graffeo, A. P. and B. L. Karger. Analysis ofindole compounds in urine by high performance liquid chromatography with fluorometric detection. Clin Chem 22: 184-187, 1976. 107. Gregorek, J. C., H. R. Seibel and R. J. Reiter. The pineal complex and its relationship to other epithalamic structures. Acta Anat 99: 425-434, 1977. 108. Halberg, F. Quo vadis basic and clinical chronobiology: promise for health maintenance. Am J Anat 168: 543-594, 1983.

FOLEY, CAIRNCROSS AND FOLDES

109. Haldar-Misra, C. and P. Pevet. The influence of different 5-methoxyindoles on the process of protein/peptide secretion as characterized by the formation of granular vesicles in the mouse pineal gland. An in vitro study. Cell Tissue Res 230: 113-126, 1983. 110. Ho, A. K. and G. M. Brown. Synchronisation of rat serum tryptophan, serotonin and N-acetylserotonin rhythms with time of feeding. J Steroid Biochem 20: 1452, 1984. 111. Ho, A. K., C. L. Chik, M. G. Joshi and G. M. Brown. Differential effects of isoproterenol injections on the levels of pineal N-acetyltransferase, serum N-acetylserotonin and melatonin. Life Sci 36: 2137-2143, 1985. 112. Hoffman, K. The influence of photoperiod and melatonin on testis size, body weight and pelage colour in the Djungarian hamster (Phodopus sungorus). J Comp Physial 85: 267-282, !973. 113. Hojo, T., A. Nakamura and Z. Tamura. Fluorometric determination of biogenic indole compounds using perchloric acid. Chem Pharm Bull (Tokyo) 30: 18%195, 1982. 114. Hojo, T., A. Nakamura and Z. Tamura. Fluorometric determination of biogenic 5-hydroxy- and 5-methyoxyindoles by high performance liquid chromatography using perchloric acid as post-column reagent. J Chromatogr 247: 157-164, 1982. 115. Howd, R. A., K. S. Seo and R. J. Wurtman. Rat liver N-acetyltransferase: inhibition by melatonin. Biochem Pharmacol 25: 977-978, 1976. 116. Iguchi, H,, K. I. Kato and H. Ibayashi. Age-dependent reduction in serum melatonin concentrations in healthy human subjects. J Clin Endocrinol Metab 55: 27-29, 1982. 117. lllnerova, H. and J. Vanecek. Effect of exposure to one minute light at night on rat pineal SNAT. Prog Brain Res 52: 241-243, 1979. 118. Illnerova, H. and J. Vanecek. The effect of light on pineal N-acetyltransferase activity. In: Current Status and Perspectives in Pineal Research--EPSG Symposium, 1985, p. 9. 119. Illnerova, H., J. Vanecek and K. Hoffman. Regulation of the pineal melatonin concentration in the rat (Rattus norvegicus) and in the Djungarian hamster (Phodopus sungorus). Comp Biochem Physiol 74A: 155-159, 1983. 120. Illnerova, H., P. Zvolsky and J. Vanecek. The circadian rhythm in plasma melatonin concentration of the urbanized man: the effect of summer and winter time. Brain Res 328: 186--189, 1985. 121. Juillard, M. T. and J. P. Collins. Pools of serotonin in the pineal gland of the mouse: the mammalian pinealocyte as a component of the diffuse neuroendocrine system. Cell Tissue Res 213: 273-291, 1980. 122. Kappers, A. A survey of advances in pineal research. In: The Pineal Gland, Vol 1: Anatomy and Biochemistry, edited by R. J. Reiter. Boca Raton, FL: CRC Press Inc., 1981, pp. 1-26. 123. Kappers, J. A. Short history of pineal discovery and research. Prog Brain Res 52: 3-22, 1979. 124. Karasek, M. and R. J. Reiter. A reciprocal relationship between the adenohypophysis and the pineal gland. Med Hypotheses 9: 1-9, 1982. 125. Kavaliers, M. Pineal mediation of the thermoregulatory and behavioral activating effects of/3-endorphin. Peptides 3: 67% 685, 1982. 126. Kavaliers, M., M. Hirst and G. C. Teskey. Ageing, opioid analgesia and the pineal gland. Life Sci 32: 227%2287, 1983. 127. Kavaliers, M., K. P. Ossenkopp and M. Hirst. Magnetic fields abolish the enhanced nocturnal analgesic response to morphine in mice. Physiol Behav 32: 261-265, 1984. 128. Kennaway, D. J., T. A. Kilmore and R. F. Seamark. Effects of melatonin implants on the circadian rhythm of plasma melatonin and prolactin in sheep. Endocrinology 110: 2186-2188, 1982. 129. King, T. S., R. W. Steger, S. Steinlechner and R. J. Reiter. Day-night differences in estimated rates of 5-hydroxytryptamine turnover in the rat pineal gland. Exp Brain Res 54: 432-436, 1984.

PINEAL INDOLES

130. King, T. S., S. Steinlechner and R. J. Reiter. Does maximal serotonin N-acetyltransferase activity necessarily reflect maximal melatonin production in the rat pineal gland? Neurosci Lett 48: 343-347, 1984. 131. Kissinger, P. T. Amperometric and coulometric detectors for high-performance liquid chromatography. Anal Chem 49: 447A-456A, 1977. 132. Kissinger, P. T., C. S. Bruntlett, G. C. Davic, L. J. Felice, R. M. Riggin and R. E. Shoup. Recent developments in the clinical assessment of the metabolism of aromatics by high performance, reversed-phase chromatography with amperometric detection. Clin Chem 23: 1449-1455, 1977. 133. Kitay, J. I. Pineal lesions and precocious puberty. A review. J Clin Endocrinol 14: 622-625, 1954. 134. Klein, D. C. Pineal gland as a model of neuroendocrine control system. In: The Hypothalamus, edited by S. Reichlin, R. J. Baldessarini and J. B. Martin. New York: Raven Press, 1978, pp. 303-329. 135. Klein, D. C., D. A. Auerbach, A. A. Namboodiri and G. H. T. Wheler. Indole metabolism in the mammalian pineal gland. In: The Pineal Gland, Vol l: Anatomy and Biochemistry, edited by R. J. Reiter. Boca Raton, FL: CRC Press Inc., 1981, pp. 199228. 136. Klein, D. C., D. A. Auerbach and J. L. Weller. Seesaw signal processing in pineal cells: homologous sensitization of adenergic stimulation of cyclic GMP accompanies homologous desensitization of/3-adrenergic stimulation of cyclic AMP. Proc Natl Acad Sci USA 78: 4625--4629, 1981. 137. Klein, D. C., G. R. Berg and J. L. Weller. Melatonin synthesis: adenosine 3',5'-monophosphate and NE stimulate NAT. Science 168: 979-980, 1970. 138. Klein, D. C., M. J. Buda, C. L. Kapoor and G. Krishna. Pineal serotonin N-acetyltransferase: abrupt decrease in adenosine 3' ,5'-monophosphate may be signal for "turnoff." Science 199: 309-311, 1978. 139. Klein, D. C. and R. Y. Moore. Pineal NAT and HIOMT: control by the retinohypothalamic tract and the suprachiasmatic nucleus. Brain Res 174: 245-262, 1979. 140. Klein, D. C., D. Sugden and J. L. Weller. Postsynaptic a-adrenergic receptors potentiate the/3-adrenergic stimulation of pineal SNAT. Proc Natl Acad Sci USA 80: 599--603, 1983. 141. Krstulovic, A. M., M. J. Friedman, P. R. Sinclair and J. Felice. Complementary use of amperometric and spectrophotometric detection for concurrent monitoring of serum tryptophan metabolites by reversed-phase liquid chromatography. Clin Chem 27: 1291-1295, 1981. 142. Kveder, S., S. Iskric and D. Keglevic. 5-Hydroxytryptophol: a metabolite of 5-hydroxytryptamine in rats. Biochem J 85: 447449, 1962. 143. Lapin, V. Pineal influence on tumor. Prog Brain Res 52: 523533, 1979. 144. Laud, C. and J. Arendt. Melatonin and biorhythms. In: Melatonin---Current Status and Perspectives, edited by N. Birau and W. Schloot. Oxford: Pergamon Press, 1981, pp. 177-182. 145. Leblanc, G. G. and R. D. Giaranello. ct-Noradrenergic potentiation of neurotransmitter-stimulated cAMP production in rat striatal slices. Brain Res 293: 57-65, 1984. 146. Le Douarin, N. M. and J. Fontaine-Perus. The neural crest and the digestive tract: developmental relationships. In: Gut Hormones, edited by S. R. Bloom and J. M. Polak. Edinburgh: Churchill Livingstone, 1981, pp. 107-118. 147. Lehman, M. N., E. L. Bittman and S. W. Newman. Role of the hypothalamic paraventricular nucleus in neuroendocrine response to daylength in the golden hamster. Brain Res 308: 25-32, 1984. 148. Leone, R. M. and R. E. Silman. Melatonin can be differentially metabolized in the rat to produce N-acetylserotonin in addition to 6-hydroxymelatonin. Endocrinology 114: 1825-1832, 1984. 149. Leong, A. S. Y. and C. D. Matthews. The pineal gland as an APUD organ: supporting evidence and implications. Med Hypotheses 5: 265-273, 1979.

289 150. Lerner, A. B. and J. D. Case. Melatonin (in Intersociety Symposium on New and Neglected Hormones). Fed Proc 19: 590593, 1960. 151. Lerner, A. B. and J. J. Nordlund. Melatonin: clinical pharmacology. J Neural Transm 13: 339-347, 1978. 152. Lewy, A. J., T. A. Wehr, F. K. Goodwin, D. A. Newsome and S. P. Markey. Light suppresses melatonin secretion in humans. Science 210: 1267-1269, 1980. 153. Lin, P. Y. T., M. C. Bulawa, P. Wong, L. Lin, J. Scott and C. L. Blank. The determination of catecholamines, indoleamines, metabolites and related enzymatic activities using three micron liquid chromatography columns J Liquid Chromatogr 7: 509538, 1984. 154. Lincoln, G. A. Differential control of luteinizing hormone and follicle-stimulating hormone by luteinizing hormone releasing hormone in the ram. J Endocrinol 80: 133-140, 1979. 155. Lincoln, G. A., M. E. Klandorf and N. Anderson. Photoperiodic control of thyroid function and wool and horn growth in rams and the effect of cranial sympathectomy. Endocrinology 107: 1543-1548, 1980. 156. Linsell, C., P. E. Mullen, R. E. Silman, R. M. Leone, M. Finney, S. J. Carter, R. J. L. Hooper, I. Smith and P. Francis. The measurement of dally fluctuations of 5-methoxytryptophol in human plasma. Prog Brain Res 52: 501-505, 1979. 157. Lowenstein, P. R. and D. P. Cardinali. Characterization of flunitrazepam and beta-carboline high affinity binding in bovine pineal gland. Neuroendocrinology 37: 150--154, 1983. 158. Lundberg, J. M., T. H6kfelt, M. Schultzberg, K. Uvn~isWaUensten, C. K6hler and S. Said. Occurrence of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in certain cholinergic neurons of the cat: evidence from combined immunohistochemistry and acetylcholinesterase staining. Neuroscience 4: 1539-1559, 1979. 159. Luo, Z. R., R. L. Schultz, E. F. Whitter and L. Vollrath. The ultrastructure of the nerve fibers and pinealogytes in the rat pineal stalk. J Pineal Res 1: 323-337, 1984. 160. Lynch, G. R. and A. L. Epstein. Melatonin-induced changes in gonads: pelage and thermogenic characters in the white-footed mouse, Peromyscus leucopus. Comp Biochem Physiol 53C: 67-68, 1976. 161. Lynch, H. J., D. C. Jimerson, Y. Ozaki, R. M. Post, W. E. Bunney, Jr. and R. J. Wurtman. Entrainment of rhythmic melatonin secretion in man to a 12 hour phase-shift in the light/ dark cycle. Life Sci 23: 1557-1563, 1978. 162. Maggi, A., D. C. U'Prichard and S. J. Enna. fl-Adrenergic regulation of c~2-adrenergic receptors in central nervous system. Science 207: 645-647, 1980. 163. Mata, M. M., B. K. Schrier, D. C. Klein, J. L. Weller and C. Y. Chia. On GABA function and physiology in the pineal gland. Brain Res 118: 383-394, 1976. 164. Matthew, E., A. G. Parfitt, D. Sugden, D. L. Engelhardt, E. A. Zimmerman and D. C. Klein. Benzodiazepines: Rat pinealocyte binding sites and augmentation of norepinephrinestimulated N-acetyltransferase activity. J Pharmacol Exp Ther 228: 434-438, 1984. 165. Maurizi, C. P. Disorder of the pineal gland associated with depression, peptic ulcers, and sexual dysfunction. South M e d J 77: 1516-1518, 1984. 166. McClung, R. and N. Dafny. Neurophysiological properties of the pineal body: II. Single unit recording. Life Sci 16: 621-628, 1975. 167. McLeod, S. D. and K. D. Cairncross. The effect of fll antagonism on oxidative deamination and N-acetylation products of pineal serotonin. Proc Aust Soc Med Res 18: 31, 1985. 168. Mefford, I. N. and J. D. Barchas. Determination of tryptophan and metabolites in rat brain and pineal tissue by reversed-phase high-performance liquid chromatography with electrochemical detection. J Chrornatogr 181: 187-193, 1980. 169. Mefford, I. N., P. Chang, D. C. Klein, M. A. A. Namboodiri, D. Sugden and J. Barchas. Reciprocal day/night relationship between serotonin oxidation and N-acetylation products in the rat pineal gland. Endocrinology 113: 1582-1586, 1983.

290 170. Mess, B. Interactions between pineal and nonreproductive endocrine glands. In: Pineal Gland and its Endocrine Role, edited by J. Axelrod, F. Fraschine and G. P. Velo. New York: Plenum Press, 1983, pp. 477-508. 171. Miline, R. The role of the pineal gland in stress. J Neural Transm 47: 191-200, 1980. 172. Mitchell, M. D., J. C. Bibby, L. Sayers, A. B. Anderson and A. C. Turnbull. Melatonin in the maternal and unbilical circulations during human parturition. Br J Obstet Gynecol 86: 2%31, 1979. 173. Meller, M. and H. W. Korf. The origin of central pineaiopetal nerve fibres in the Mongolian gerbil as demonstrated by the retrograde transport of horseradish peroxidase. Cell Tissue Res 230: 273-287, 1983. 174. Morita, 1., T. Masujima, H. Yoshida and H. Imai. Enrichment and high-performance liquid chromatography analysis of tryptophan metabolites in plasma. Anal Biochem 118: 142-146, 1981. 175. Morton, D. J. and B. Potgieter. Relationships between methoxyindoles and hydroxyindoles formed from 5-hydroxytryptamine in rat pineal gland. J Endocrinol 95: 253-256, 1982. 176. Namboodiri, M. A. A., D. Sugden, D. C. Klein, R. Grady and I. N. Mefford. Rapid nocturnal increase in ovine pineal N-acetyltransferase activity and melaton in synthesis: effects of cycloheximide. J Neurochem 45: 832-835, 1985. 177. Namboodiri, M. A. A., D. Sugden, D. C. Klein, L. Tamarkin and I. N. Mefford. Serum melatonin and pineal indoleamine metabolism in a species with a small day/night N-acetyltransferase rhythm. Comp Biochem Physiol 80B: 731-736, 1985. 178. Namboodiri, M. A. A., J. L. Weller and D. C. Klein. Evidence of inactivation of rat pineal SNAT by protein thioldisulfide exchange. J Biol Chem 255: 6032-6035, 1980. 179. Narasimhachari, N., E. Kemspter and M. Anbar. 5-Methoxytryptamine in rat hypothaiamus and human CSF: A fact or artifact? Biomed Mass Spectrom 7: 231-235, 1980. 180. Nieuwenhuis, J. J. Arginine vasotocin (AVT), an alleged hormone of the mammalian pineal gland. Life Sci 35: 1713-1724, 1984. 181. Niles, L. P., G. M. Brown and L. J. Grota. Endocrine effects of the pineal gland and neutralization of circulating melatonin and N-acetylserotonin. Can J Physiol Pharmacol 55: 537-544, 1977. 182. Niles, L. P., G. M. Brown and L. J. Grota. Characteristics of high affinity binding of [3H]-acetylserotonin in rat brain. Neuropharmacology 22: 1311-1314, 1983. 183. Niles, L. P., G. M. Brown and R. K. Mishra. A serotoninergic component of [3H]N-acetyl serotonin binding in mammalian brain. Prog Neuropsychopharmacol Biol Psychiatry 6: 421424, 1982. 184. Niles, L. P., G. M. Brown and R. K. Mishra. Effects of blinding and pinealectomy on regional brain mooamine concentrations. J Neurosci Res 10: 53-60, 1983. 185. Nir, I. and N. Hirschmann. Effect of psychotropic drugs on [14C] tryptophan metabolism in cultured rat pineal gland. Biochem Pharmacol 32: 213%2141, 1983. 186. Nir, I., N. Hirschmann and F. G. Sutman. Inhibition of pineal HIOMT by pyridoxal-6-phosphate. Biochem Pharmacol 25: 581-583, 1976. 187. Oxenkrug, G. F., R. McCauley, I. M. Mclntyre and C. Filipowicz. Selective inhibition of MAO-A but not MAO-B activity increases rat pineal melatonin. J Neural Transm 61: 265-270, 1985. 188. Pang, S. F., M. K. Yip, H. W. Lin, G. M. Brown and H. W. Tsui. Diurnal rhythms of immunoreactive N-acetyl serotonin and melatonin in the serum of male rats. Acta Endocrinol 95: 571-576, 1980. 189. Pang, S. F., H. S. Yu and P. L. Tang. A diurnal rhythm of N-acetylserotonin in the retina of rats. Neurosci Lett 21: 197200, 1981.

FOLEY, CAIRNCROSS AND FOLDES 190. Parfitt, A. G. and D. C. Klein. Sympathetic nerve endings in the pineal gland protect against acute stress-induced increase in N-acetyltransferase (EC 2.3.1.5) activity. Endocrinology 99: 840-851, 1976. 191. Parkington, H. C., I. McCance and H. A. Coleman. Electrophysiological evidence for a significant central input to the pineal in the guinea-pig. Proc Aust Physiol Pharmacol Soc 16: 16P, 1985. 192. Parkington, H. C., I. McCance and H. A. Coleman. Interactions between effects mediated by t~- and fl-adrenoceptor activation in cells of the guinea-pig pineal. Proc Aust Physiol Pharmacol Soc 16: 204P, 1985. 193. Pearse, A. G. E. Peptides in brain and intestine. Nature 262: 92-94, 1976. 194. Pearse, A. G. E. The APUD concept and its relationship to the neuropeptides. In: Brain Peptides: A New Endocrinology, edited by A. M. Gotto, E. J. Peck and A. E. Boyd. Amsterdam: Elsevier/North Holland Biomedical Press, 1979, pp. 8%101. 195. Pelayo, F., M. L. Dubocovich and S. z. Langer. Possible role of cyclic nucleotides in regulation of NA release from rat pineal through presynaptic adrenoreceptors. Nature 274: 76-78, 1978. 196. P6vet, P. Peptides in the pineal gland of vertebrates. Ultrastructural, histochemical, immunocytochemical and radioimmunological aspects. In: The Pineal Organ: Photobiology. Biochronometry, Endocrinology, edited by A. Oksche and P. P6vet. Amsterdam: Elsevier/North Holland Biomedical Press, 1981, pp. 211-236. 197. P6vet, P. The different classes of proteic and peptidic substances present in the pineal gland. In: The Pineal Gland and its Endocrine Role, edited by J. Axelrod, F. Fraschini and C. P. Velo. New York: Plenum Press, 1983, pp. 113-145. 198. P6vet, P. Is 5-methoxytryptamine a pineal hormone? Psychoneuroendocrinology 8: 61-73, 1983. 199. P6vet, P., M. G. M. Balemans, W. C. Legerstee and B. Vivien-Roels. Circadian rhythmicity of the activity of hydroxyindole-O-methyl transferase (HIOMT) in the formation of melatonin and 5-methoxytryptophol in the pineal, retina and harderian gland of the golden hamster. J Neural Transm 49: 229-245, 1980. 200. Pilc, A. and S. J. Enna. Synergistic interaction between ~- and /3-adrenergic receptors in rat brain slices: possible site for antidepressant drug action. Life Sci 37:1183-1194, 1985. 201. Plotka, E. D., U. S. Seal and L. J. Verme. Morphologic and metabolic consequences of pinealectomy in deer. In: The Pineal Gland, Vo1111: Extra-Reproductive Effects, edited by R. J. Reiter. Boco Raton, FL: C.R.C. Press Inc., 1982, pp. 107152. 202. Prechel, M. M., T, K. Audhya and D. H. Schlesinger. A seasonal variation in arginine vasotocin immunoreactivity in rat pineal glands. Endocrinology 112: 1474-1478, 1983. 203. Prechel, M., T. K. Audhya and W. H. Simmons. Pineal arginine vasotocin activity increases 200-fold during August in adult rats and hamsters. J Pineal Res 1: 175-180, 1984. 204. Preslock, J. P. The pineal gland: basic implications and clinical correlations. Endocr Rev 5: 282-308, 1984. 205. Quay, W. B. Retrograde perfusions of the pineal region and the question of pineal vascular routes to brain and choroid plexuses. Am J Anat 137: 387-402, 1973. 206. Quay, W. B. General biochemistry of the pineal gland of mammals. In: The Pineal Gland, Vol 1: Anatomy and Biochemistry, edited by R. J. Reiter. Boca Raton, FL: CRC Press Inc., 1981, pp. 173-198. 207. Quay, W. B. and K. G. Gorray. Pineal effects on metabolism and glucose homeostasis: evidence for lines of humoral mediation of pineal influences on tumor growth. J Neural Transm 47: 107-120, 1980. 208. Ralph, C. L., B. T. Firth, W. A. Gem and D. W. Owens. The pineal complex and thermoregulation. Biol Rev 54:41-72, 1979. 209. Reiter, R. J. Comparative physiology: pineal gland. Annu Rev Physiol 35: 305-328, 1973. 210. Reiter, R. J. Pineal and associated neuroendocrine rhythms. Psychoneuroendocrinology 1: 255-263, 1976.

PINEAL INDOLES 211. Reiter, R. J. (Ed.). lne Pineal Gland, Vol I: Anatomy and Biochemistry. Boca Raton. FL: CRC Press Inc., 1981. 212. Reiter, R. J. (Ed.). The Pineal Gland, Vol III: ExtraReproductive Effects. Boca Raton, FL: CRC Press Inc., 1982. 213. Reiter, R. J. The pineal gland: an intermediary between the environment and the endocrine system. Psychoneuroendocrinology 8: 31-40, 1983. 214. Reiter, R. J., H. C. Hurlbut, G. L. Brainard, S. Steinlechner and B. A. Richardson. Influence of light irradiance on hydroxyindole-O-methyl transferase activity, serotonin-Nacetyltransferase activity, and radioimmunoassayable melatonin levels in the pineal gland of the diurnally active Richardson's ground squirrel. Brain Res 292: 151-157, 1983. 215. Reiter, R. J., A. J. Lukaszyk, M. K. Vaughan and D. E. Blask. New horizons of pineal research. Am Zool 16: 93-101, 1976. 216. Reiter, R. J. and M. K. Vaughan. Pineal antigonadotrophic substances: polypeptides and indoles. Life Sci 31: 159-171, 1981. 217. Reuss, S., J. Olcese and L. Vollrath. Electrical stimulation of the hypothalamic paraventricular nuclei inhibits pineal melatonin synthesis in male rats. Neuroendocrinology 41: 192-196, 1985. 218. Reuss, S., P. Semm and L. Vollrath. Electrophysiological investigations on the central innervation of the rat and guinea-pig pineal gland. J Neural Transm 60: 31-43, 1984. 219. Reuss, S., P. Semm and L. Vollrath. Changes in the electrical activity of the rat pineal gland following stimulation of the cervical sympathetic ganglia. J Auton Nerv Syst 62: 281-288, 1985. 220. Reuss, S. and L. Vollrath. Electrophysiological properties of rat pinealocytes: evidence for circadian and ultradian rhythms. Exp Brain Res 55: 455--461, 1984. 221. Ritta, M. N. and D. P. Cardinali. Involvement of a-receptors in norepinephrine-induced prostaglandin E2 release by rat pineal gland in vitro. Neurosci Lett 31: 307-311, 1981. 222. Ritta, M. N., D. P. Cardinali and M. I. Keller Sarmiento. Prostaglandin E2 increases adenosine 3',5'-monophosphate concentration and binding site occupancy and stimulates SNAT activity in pineal glands in vitro. Mol Cell Endocrinol 23: 151-159, 1981. 223. RoUag, M. D. Methods for measuring pineal hormones. In: The Pineal Gland. Vol 1: Anatomy and Biochemistry, edited by R. J. Reiter. Boca Raton, FL: CRC Press Inc., 1982, pp. 273-302. 224. Rollag, M. D. and G. D. Niswender. Radioimmunoassay of serum concentrations of melatonin in sheep exposed to different lighting regimens. Endocrinology 98: 482-489, 1976. 225. Romijn, H. J. The pineal, a tranquillizing organ? Life Sci 23: 2257-2274, 1978. 226. R0nnekliev, O. K., M. J. Kelly, M. M011er and W. Wuttke. Electrophysiological and morphological evidence of direct central innervation of the pineal gland. Pfliigers Arch Suppl 373: Abstract 187, 1978. 227. Rcnnekleiv, O. K., M. J. Kelly and W. Wuttke. Single unit recordings in the rat pineal gland: evidence for habenulo-pineal neural connections. Exp Brain Res 39" 187-192, 1980. 228. Ronnekleiv, O. K. and M. M~ller. Brain-pineal nervous connections in the rat: an ultrastructural study following habenular lesion. Exp Brain Res 37: 551-562, 1979. 229. Rowe, V. and J. Parr. Developmental changes in the stimulation of SNAT activity and melatonin synthesis in rat pineal in monolayer culture. J Pharmacol Exp Ther 218: 97-102, 1981. 230. Seggie, J., L. Campbell, G. M. Brown and L. J. Grota. Melatonin and N-acetylserotonin stress responses: effects of type of stimulation and housing. J Pineal Res 2: 39--49, 1985. 231. Semm, P. Neurobiological investigations on the magnetic sensitivity of the pineal gland in rodents and pigeons. Comp Biochem Physiol 76A: 683-689, 1983. 232. Semm, P. Electrophysiology of the mammalian pineal gland: evidence for rhythmical and nonrhythmical elements and for magnetic influence on electrical activity. In: Vertebrate Circadian Systems, edited by J. Aschoff, S. Daan and G. A. Groos. Berlin: Springer Verlag, 1983, pp. 147-157.

291 233. Serum, P., T. Schneider and L. Vollrath. Effects of an earthstrength magnetic field on electrical activity of pineal cells. Nature 288: 607-608, 1980. 234. Serum, P. and L. Vollrath. Electrophysiological properties of single cells of the guinea pig epiphysis cerebri. Pfliigers Arch Suppl 373: Abstract 188, 1978. 235. Serum, P. and L. Vollrath. Electrophysiology of the guinea-pig pineal organ: sympathetically influenced cells responding differently to light and darkness. Neurosci Lett 12: 93-96, 1979. 236. Semm, P. and L. Vollrath. Alterations in the spontaneous activity of cells in the guinea pig pineal gland and visual system produced by pineal indoles. J Neural Transm 53: 265-275, 1982. 237. Serum, P. and L. Vollrath. Electrical responses of homing pigeon and guinea-pig Purkinje cells to pineal indoleamines applied by microelectrophoresis. J Comp Physiol 154: 675-681, 1984. 238. Shibuya, H., M. Torn and S. Watanabe. A circadian rhythm of tryptophan hydroxylase in the pineal gland of the rat. Brain Res 138: 364-368, 1978. 239. Silman, R. 5-Methoxyindoles in man. In: The Pineal Organ: Photobiology, Biochronometry, Endocrinology, edited by A. Oksche and P. P~vet. Amsterdam: Elsevier/North Holland Biomedical Press, 1981, pp. 335-344. 240. Sitaram, B. R.and G. J. Lees. Diurnal rhythm and turnover of tryptophan hydroxylase in the pineal gland of the rat. J Neurochem 31: 1021-1026, 1981. 241. Sitaram, B. R. and G. J. Lees. Effect of oxygen on the induction of tryptophan hy0xgxvlase by adrenergic agents in organ cultures of rat pineal glands. J Neurochem 42:1183-1185, 1984. 242. Smith, I. Indoles of pineal origin: biochemical and physiological status. Psychoneuroendocrinology 8: 41-60, 1983. 243. Smith, J. A. The biochemistry and pharmacology of melatonin. In: Melatonin---Current Status and Perspectives, edited by N. Biran and W. Schloot. Oxford: Pergamon Press, 1981, pp. 135-141. 244. Smith, I. D., D. L. Larson Carter, C. A. Laud, R. M. Leone, R. E. Silman, S. J. Carter, P. Francis, P. E. Muilen, R. J. L. Hooper and M. D. A. Finnie. O-acetyl-5-methoxytryptopholtentative identification in pineal glands. Prog Brain Res 52: 259-261, 1979. 245. Smyth, G. A. and L. Lazarus. Growth hormone regulation by melatonin and serotonin. Nature 244: 230-231, 1973. 246. Smyth, G. A. and L. Lazarus. Suppression of human growth hormone secretion by melaionin and cyproheptadine, j C-lin Invest 54: 116--121, 1974. 247. Steinlechner, S., T. H. Champney, M. L. Houston and R. J. Reiter. Simultaneous determination of N-acetyltransferase activity, hydroxyindole-O-methyl-transferase activity, and melatonin content in the pineal gland of the Syrian hamster. Proc Soc Exp Biol Med 175: 93-97_ 1984. 248. Stulik, K. and V. Pacakova. Electrochemical detection in high performance liquid chromatography. CRC Crit Rev Anal Chem 14: 297-351, 1984. 249. Sugden, D. and A. Fletcher. Changes in the rat sleep-wake cycle produced by D,L-6-fluorotryptophan. a competitive inhibitor of tryptophan hydroxylase. Psychopharmacology (Berlin) 74: 369-373, 1981. 250. Sugden, D. and D. C. Klein. Regulation of rat pineal HIOMT in neonatal and adult rats. J Neurochem 40: 1647-1653, 1983. 251. Sugden, D. and D. C. Klein. fl-Adrenergic receptor control of rat pineal HIOMT. Endocrinology 113: 348--353, 1983. 252. Sugden, D. and D. C. Klein. Inactivation of rat pineal hydroxyindole-O-methyltransferase by disulfides: evidence for an endogenous inactivator. J Steroid Biochem 20: 1469, 1984. 253. Sugden, D., M. A. A. Namboodiri, D. C. Klein, R. K. Grady and I. N. Mefford. Ovine pineal indoles: effects of trVntg_vhan or L-5-hydroxytryptophan administration. J urochem 44: 769-772, 1985.

292

254. Sugden, D., M. A. A. Namboodiri, D. C. Klein, J. E. Pierce, R. Grady and I. N. Mefford. Ovine pineal cq-adrenoceptors: characterization and evidence for a functional role in the regulation of serum melatonin. Endocrinology 116: 1960-1967, 1985. 255. Sugden, D., J. Vanecek, D. C. Klein, T. P. Thomas and W, B. Anderson. Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes. Nature 314: 35%361, 1985. 256. Sugden, D., J. L. Weller, D. C. Klein, K. L. Kirk and C. R. Creveling. Alpha-adrenergic potentiation of beta-adrenergic stimulation of rat pineal N-acetyltransferase: studies using cirazoline and fluorine analogs of noreprephrine. Biochem Pharmacol 33: 3947-3950, 1984. 257. Taborsky, R. G. 5-Hydroxytryptophol: evidence for its having physiological properties. Experientia 27: 92%930, 1971. 258. Tamarkin, L., P. Abastillas, H. C. Chen, A. McNemar and J. B. Sidbury. The daily profile of plasma melatonin in obese and Proder-Willi syndrome children. J Clin Endocrinol Metab 55: 491-495, 1982. 259. Tamarkin, L., C. J. Baird and O. F. X. Almeida. Melatonin: A coordinating signal for mammalian reproduction? Science 227: 714-720, 1985. 260. Tang, P. and S. F. Pang. Effect of N-acetylserotonin on the serum level of thyroid-stimulating hormone in the male rat. Neuroendocrinology 36: 268-271, 1983. 261. Tapp, E. The pineal gland in malignancy. In: The Pineal Gland. Vol 111: Extra-Reproductive Effects, edited by R. J. Reiter. Boca Raton, FL: CRC Press Inc., 1982, pp. 171-188. 262. Turek, F. W. Are the suprachiasmatic nuclei the location of the biological clock in mammals? Nature 292: 28%290, 1981. 263. Urry, R. L., K. A. Dougherty, J. L. Frenn and L. C. Ellis. Factors other than light affecting the pineal gland: hypophysectomy, testosterone, dihydrotestosterone, estradiol, cryptorchidism and stress. A m Zool 16: 79-91, 1976. 264. Vacas, M. I., M. I. K. Sarmiento and D, P. Cardinali. Interaction between a- and/3-adrenoceptors in rat pineal adenosine cyclic Y,5'-monophosphate phosphodiesterase activation. J Neural Transm 26: 295-304, 1985. 265. Vanecek, J. and H. Illnerova. Changes of rhythm in rat SNAT following a one minute light pulse at night. Prog Brain Res 52: 245-248, 1979. 266. Vanecek, J., D. Sugden, J. Weller and D. C. Klein. Atypical synergistic al- and /3-adrenergic regulation of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in rat pinealocytes. Endocrinology 116: 2167-2173, 1985. 267. Vassilieff, V. S., T. L. Saunkes and G. M. Anderson. Superior cervical ganglionectomy: effect on indolic compounds in rat pineal gland. J Neural Transm 55: 45-52, 1982. 268. Vaughan, G. M., R. Bell and A. de la Pena. Nocturnal plasma melatonin in humans: episodic pattern and influence of light. Neurosci Lett 14: 81-84, 1979. 269. Verbiese-Genard, N., M. Hanocq, C. Alvoet and L. Molle. Degradation study of catecholamines, indoleamines and some of their metabolites in different extraction media by chromatography and electrochemical detection. Anal Biochem 134: 170-175, 1983. 270. Vickrey, T. M. (Ed.). Liquid Chromatography Detectors. New York: Marcel Decker Inc., 1983. 271. Volle, R. L. and L. F. Quenzer. Regulation of cyclic GMP levels in nerve tissue. Fed Proc 42: 309%3102, 1983. 272. Vollrath, L. Handbuch der Mikroskopischen Anatomie des Menschen/7: The Pineal Organ. Berlin: Springer Verlag, 1981. 273. Vollrath, L., A. Kantarjian and C. Howe. Mammalian pineal gland: 7 day rhythmic activity? Experientia 31: 458--460, 1975. 274. Vriend, J. Testing the TRH hypothesis of pineal function. Med Hypotheses 4: 376-387, 1978. 275. Waldhauser, F., H. Frisch, M. Waldhauser, G. Weiszenbacher, U. Zeithuber and R. J. Wurtman. Fall in nocturnal serum melatonin during prepuberty and pubescence. Lancet 1: 362-365, 1984.

FOLEY, CAIRNCROSS AND FOLDES 276. Walker, R. F. and V. J. Aloya. NE stimulates serotonin secretion from rat pineal glands. Brain Res 343: 188-190, 1985. 277. Webb, S. M., T. H. Champney, A. K. Lewinski and R. J. Reiter. Photoreceptor damage and eye pigmentation: influence on the sensitivity of rat pineal N-acetyltransferase activity and melatonin levels to light at night. Neuroendocrinology 40: 205-209, 1985. 278. Webley, G. E., H. Mehl and K. P. Willey. Validation of a sensitive direct assay for melatonin for investigation of circadian rhythms in different species. J Endocrinol 106: 387-394, 1985. 279. Weinberg, U., R. D. D'Eletto, E. D. Weitzman, S. Erlich and C. S. Hollander. Circulating melatonin in man: episodic secretion throughout the light-dark cycle. J Clin Endocrinol Metab 48: 114-118, 1979. 280. Welker, H. A., P. Semm, R. P. Willig, J. C. Commentz, W. Willschko and L. Vollrath. Effects of an artificial magnetic field on serotonin N-acetyltransferase activity and melatonin content of the rat pineal gland. Exp Brain Res 50: 426-432, 1983. 281. Welker, H. A. and L. Vollrath. The effects of a number of short-term exogenous stimuli on pineal serotonin N-acetyltransferase activity in rats. J Neural Transm 59: 60-80, 1984. 282. Wetterberg, L. The relationship between the pineal gland and the pituitary-adrenal axis in health, endocrine and psychiatric conditions. Psychoneuroendocrinology 8: 75-80, 1983. 283. Wetterberg, L., B. Aperia, J. Beck-Fris, B. F. Kjellman, J. G. Ljunggren, A. Nilsonne, U. Petterson, A. Tham and F. Under. Melatonin and cortisol levels in psychiatric illness. Lancet 2: 100, 1982. 284. Wetterberg, L. and O. Eriksson. Melatonin in human serum--a collaborative study of current radioimmunoassays. In: Melatonin: Current Status and Perspectives, edited by N. Birand and W. Schloot. Oxford: Pergamon Press, 1980, pp. 15-20. 285. Wheler, G. H. T. and D. C. Klein. Taurine release from the pineal gland is stimulated via a/3-adrenergic mechanism. Brain Res 187: 155-164, 1980. 286. White, B. H., K. Mosher and S. Binkley. Rat pineal N-acetyltransferase activity: stimulation, exhaustion and recovery. Endocrinology 117: 1050-1056, 1985. 287. White, B. H., K. Mosher and S. Binkley. Phase shift of daily profiles of N-acetyltransferase in the rat pineal gland. J Pineal Res 2: 201-208, 1985. 288. Wiechmann, A. F., D. Bok and J. Horwitz. Localization of hydroxyindole-O-methyltransferase in the mammalian pineal gland and retina. Invest Ophthamol Vis Sci 26: 253-265, 1985. 289. Williams, A. H. Some observations on the involvement of the pineal gland in the control of natural wool-shedding by Wiltshire horn sheep. In: Proceedings o f Second National Conferenee on Wool Harvesting Research and Development, edited by P. R. W. Hudson. Sydney: Australian Wool Corporation, 1981, pp. 37-40. 290. Wilson, B. W., L. E. Anderson, D. I. Hilton and R. D. Phillips. Chronic exposure to 60-Hz electric fields: effects on pineal function in the rat. Bioelectromagnetics 2: 371-380, 1981. 291. Wilson, B. W., H. J. Lynch and Y. Ozaki. 5-Methoxytryptophol in rat semen and pineal: detection, quantitation and evidence for daily rhythmicity. Life Sci 23: 101%1024, 1978. 292. Wurtman, R. J. and H. B. Lieberman. Melatonin secretion as a mediator of circadian variations in sleep and sleepiness. J Pineal Res 2: 301-303, 1985. 293. Yamada, Y., Y. Sugimoto and K. Horisaka. Simultaneous determination of tryptophan and its metabolites in mouse brain by high-performance liquid chromatography with fluorometric detection. Anal Biochem 129: 460--463, 1982. 294. Yang, H.-Y. T., C. Goridis and N. H. Neff. Properties of monoamine oxidases in sympathetic nerve and pineal gland. J Neurochem 19: 1241-1250, 1972.

PINEAL INDOLES 295. Young, S. N. ana G. M. Anderson. Factors influencing melatonin, 5-hydroxytryptophol, 5-hydroxyindoleacetic acid, 5-hydroxytryptamine and tryptophan in rat pineal glands. Neuroendocrinology 35: 464--468, 1982. 296. Young, I. M., R. M. Leone, P. Francis, P. Stovell and R. E. Silman. Melatonin is metabolized to N-acetyl serotonin and 6-hydroxymelatonin in man. J Clin Endocrinol Metab 60:114.119, 1985. 297. Yuwiler, A. Vasoactive intestinal peptide stimulation of pineal serotonin-N-acetyl transferase activity: general characterisitics. J Neurochem 41: 146--153, 1983. 298. Zatz, M. Pharmacology of rat pineal gland. In: The Pineal Gland. Vol 1. Anatomy and Biochemistry, edited by R. J. Reiter. Boca Raton, FL: CRC Press Inc., 1982, pp. 225-242. 299. Zatz, M. Phorbol esters mimic ct-adrenergic potentiation of serotonin N-acetyltransferase induction in the rat pineal. J Neurochem 45: 637-639, 1985.

293 300. Zatz, M. The role of cyclic nucleotides in the pineal gland. In: Cyclic Nucleotides. Part H: Physiology and Pharmacology, edited by J. W. Kebabian and J. A. Nathanson. Berlin: Springer Verlag, 1982, pp. 691-710. 301. Zatz, M. and M. J. Brownstein. Intraventricular carbachol mimics the effect of light on the circadian rhythm in rat pineal gland. Science 203: 358-361, 1979. 302. Zatz, M. and M. Weinstock. Electric field stimulation releases norepinephrine and cyclic GMP from the rat pineal gland. Life Sci 22: 767-772, 1978. 303. Zigmond, R. E., C. Baldwin and C. W. Bowers. Rapid recovery of function after partial denervation of the rat pineal gland suggests a novel mechanism for neural plasticity. Proc Natl Acad Sci USA 78: 3959-3963, 1981.