Proefschrift - Utrecht / Summary in English.

Preface: The general indications that the pineal gland might be involved in homeostasis, and more specifically the evidence suggesting a role in amelioration of seizure states warranted further investigation . No reports had examined a possible link between anticonvulsant drug administration and pineal gland function, and few enabled any type of presumption to be made as to possible effects. This study was an attempt to evaluate in which ways anticonvulsant drugs might alter pineal gland indole metabolism, with a view to increasing understanding of the role of the pineal in modulation of epileptic discharges. In order to make the study as meaningful as possible extensive preliminary investigations were necessary. Pharmacokinetic determinations gave an indication of tissue concentrations of the drugs, which could then be related to observed effects. As far as possible, where existing information was lacking, the catalytic behaviour of the various enzymes was characterised in order to explain any observed effects at a molecular level. An attempt was also made to characterise the regulatory mechanisms controlling indole metabolism, again in order to define the pharmacological effects exerted by the drugs used. The complexity of the system made it impossible to suggest a single uniform regulatory hypothesis, although some significant observations were made. Finally, the studies involving the anticonvulsant drugs were conducted on intact animals, isolated organs and individual enzymes in an attempt to determine whether the observed effects were occuring at a molecular, local or central level.

An attempt was made to use the pineal gland as a model for the study of the primary mode of action of sympathoactive agents. Two drugs were investigated, viz. alpha-methyldopa and ephedrine whose mode of action is not entirely clear. Organ cultures of pineal glands from rats treated chronically with alpha-methyldopa showed enhanced conversion of radioactive serotonin to melatonin (aMT) , as well as its precursor Nacetylserotonin (aHT). This treatment was also found to raise Nacetyltransferase (NAT) activity. These increases associated with alpha-methyldopa treatment were further enhanced by the beta-adrenergic agonist, isoproterenol, suggesting a supersensitivity-type effect occurring at the level of the beta-receptor. A subsequent binding study, however, showed a decrease in beta-receptor binding with exposure to alpha-methyldopa, providing mitigating evidence against the occurrence of a supersensitivity phenomenon. It is possible that a metabolite of alpha-methyldopa acts as an alpha 1 and beta-adrenergic agonist, resulting in greater melatonin (aMT) and N-acetylserotonin (aHT) synthesis than by a beta-adrenergic agonist, isoproterenol. Combined treatment of pineals with alpha-methyldopa and an alphareceptor blocker, phentolamine, resulted in melatonin (aMT) , Nacetylserotonin (aHT) , and N-acetyltransferase (NAT) activity levels which were lower than those obtained with alpha-methyldopa treatment alone, thus confirming the alpha-adrenergic activity of the metabolite of alpha-methyldopa. Additional pineal metabolites were isolated and measured simultaneously in the organ culture experiments. Organ cultures of rat pineal glands treated with ephedrine showed raised levels of melatonin (aMT) and N-acetylserotonin (aHT). Treatment with ephedrine also produced raised N-acetyltransferase activity. A further enhancement of these parameters was induced by norepinephrine, suggesting a supersensitivity-type effect occurring at the level of the beta-adrenergic receptor. Rats were treated with reserpine (a norepinephrine depleter) and the pineals exposed to ephedrine. Endogenous norepinephrine normally released by the action of ephedrine was thus absent, and under these conditions, levels of melatonin (aMT) and N-acetylserotonin (aHT) were reduced. N-acetyltransferase (NAT) activity was also reduced, but maintained levels pointing to substantial adrenergic activity of ephedrine as well as norepinephrine released by virtue of the drug's action. A subsequent binding study showed a decrease in beta-adrenergic receptor binding with exposure to ephedrine and a further decrease in ephedrine treated pineals from reserpine treated rats, thus ruling out the occurrence of a supersensitivity phenomenon. It is possible that both ephedrine and released norepinephrine have alpha- and beta-receptor activity. Additional pineal metabolites were isolated and measured in the organ culture experiments. A 16-hour time profile of the production of melatonin (aMT) and N-acetylserotonin (aHT) with norepinephrine and ephedrine treatment provided useful information regarding the course of action of the two agents. A pineal cell-culture system was developed and exposed to ephedrine and norepinephrine. N-acetyltransferase (NAT) activity levels measured after exposure to these agents were raised, confirming the adrenergic activity of both in the model. Finally, an HPLC system coupled to a UV detector was used in an attempt to measure melatonin (aMT) extracted from pineal organ culture media. The results showed that melatonin could be measured by this method, however, a more sensitive detection system was recommended for future work

An attempt was made to use the pineal gland as a model for the study of the primary mode of action of sympathoactive agents. Two drugs were investigated, viz. alpha-methyldopa and ephedrine whose mode of action is not entirely clear. Organ cultures of pineal glands from rats treated chronically with alpha-methyldopa showed enhanced conversion of radioactive serotonin to melatonin (aMT) , as well as its precursor Nacetylserotonin (aHT). This treatment was also found to raise Nacetyltransferase (NAT) activity. These increases associated with alpha-methyldopa treatment were further enhanced by the beta-adrenergic agonist, isoproterenol, suggesting a supersensitivity-type effect occurring at the level of the beta-receptor. A subsequent binding study, however, showed a decrease in beta-receptor binding with exposure to alpha-methyldopa, providing mitigating evidence against the occurrence of a supersensitivity phenomenon. It is possible that a metabolite of alpha-methyldopa acts as an alpha 1 and beta-adrenergic agonist, resulting in greater melatonin (aMT) and N-acetylserotonin (aHT) synthesis than by a beta-adrenergic agonist, isoproterenol. Combined treatment of pineals with alpha-methyldopa and an alphareceptor blocker, phentolamine, resulted in melatonin (aMT) , Nacetylserotonin (aHT) , and N-acetyltransferase (NAT) activity levels which were lower than those obtained with alpha-methyldopa treatment alone, thus confirming the alpha-adrenergic activity of the metabolite of alpha-methyldopa. Additional pineal metabolites were isolated and measured simultaneously in the organ culture experiments. Organ cultures of rat pineal glands treated with ephedrine showed raised levels of melatonin (aMT) and N-acetylserotonin (aHT). Treatment with ephedrine also produced raised N-acetyltransferase activity. A further enhancement of these parameters was induced by norepinephrine, suggesting a supersensitivity-type effect occurring at the level of the beta-adrenergic receptor. Rats were treated with reserpine (a norepinephrine depleter) and the pineals exposed to ephedrine. Endogenous norepinephrine normally released by the action of ephedrine was thus absent, and under these conditions, levels of melatonin (aMT) and N-acetylserotonin (aHT) were reduced. N-acetyltransferase (NAT) activity was also reduced, but maintained levels pointing to substantial adrenergic activity of ephedrine as well as norepinephrine released by virtue of the drug's action. A subsequent binding study showed a decrease in beta-adrenergic receptor binding with exposure to ephedrine and a further decrease in ephedrine treated pineals from reserpine treated rats, thus ruling out the occurrence of a supersensitivity phenomenon. It is possible that both ephedrine and released norepinephrine have alpha- and beta-receptor activity. Additional pineal metabolites were isolated and measured in the organ culture experiments. A 16-hour time profile of the production of melatonin (aMT) and N-acetylserotonin (aHT) with norepinephrine and ephedrine treatment provided useful information regarding the course of action of the two agents. A pineal cell-culture system was developed and exposed to ephedrine and norepinephrine. N-acetyltransferase (NAT) activity levels measured after exposure to these agents were raised, confirming the adrenergic activity of both in the model. Finally, an HPLC system coupled to a UV detector was used in an attempt to measure melatonin (aMT) extracted from pineal organ culture media. The results showed that melatonin could be measured by this method, however, a more sensitive detection system was recommended for future work.

The purpose of this project was to investigate the role of melatonin in the pathophysiology of anxiety in humans. The literature study confirmed the intimate relationship between serotonin and melatonin. Melatonin is not only able to act as an agonist (in physiological concentrations) and an antagonist (at higher concentrations) on serotonin receptors but via control of brain pyridoxal kinase activity might have an effect on GABA, serotonin, dopamine and norepinephrine synthesis. A clinical trial to investigate melatonin's effect on anxiety in humans was conducted as a pilot study. Thirty patients complaining of anxiety participated in a liN of 1" double blind placebo controlled trial. During the experiment each subject was thus exposed to melatonin and a placebo for a week at a time on two occasions. During the first phase of the experiment, (Pair '1) patients showed a statistically significant reduction in their anxiety levels during the first period (P1P1), which was not the case during the second period (P1P2). The improvement however continued during the second phase of the experiment (Pair 2) so that there was also a statistically significant improvement during P 2 P 2 (Period 2 / Pair 2) when placebo was administered. It could not conclusively be shown that melatonin was responsible for the improvement in the patients' anxiety. The explanation for these results suggests thelt the improvement was due to a: 1) placebo effect throughout, 2) psychotherapeutic effect due to contact with a clinician, 3) melatonin induced phase shift in the patient's endogenous melatonin response curve, 4) combination of all 3 options. This pilot study lays the groundwork for a much more exhaustive study in which the melatonin of the patients is determined before melatonin is administered, the role of the clinician is clarified and the most appropriate time for melatonin administration is sought .

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The multiple functions of the pineal gland have been collectively interpreted as constituting a general anti-stressogenic role. The adrenal glands play a central role in maintaining homeostasis. The major neuroendocrine consequence of long-term stress is elevated circulating glucocorticoid levels. In this study, the effect of chronic, oral hydrocortisone treatment on pineal biochemistry was investigated in male Wi star rats of the albino strain. The results show that seven days of oral hydrocortisone treatment endows the pineal gland with the ability to increase melatonin synthesis in organ culture. The increase is accompanied by a rise in NAT activity, cyclic AMP levels and enhanced specific binding to the pineal B-adrenergic receptors. It appears that hydrocortisone sensitizes the pineal gland to stimulation by B-adrenergic agonists. thus rendering the pineal more responsive to B-adrenergic agonists. Further studies were directed at demonstrating an anti-stressogenic function for the pineal gland by investigating whether the principal pineal indole, melatonin. could protect against the deleterious effects of elevated. circulating drocortisone levels. The results show that chronic, oral hydrocortisone treatment significantly increases liver tryptophan pyrrolase activity. The catabolism of tryptophan by tryptophan pyrrolase is an important determinant of tryptophan availability to the brain, and therefore, brain serotonin levels. The findings show that melatonin inhibits basal and hydrocortisone-stimulated liver tryptophan pyrrolase apoenzyme activity in a dose-dependent manner. This inhibition suggests that melatonin may protect against excessive loss of tryptophan from circulation and against deficiencies in the cerebral serotinergic system which are associated with mood and behavioural disorders. It was shown that another deleterious effect of chronic hydrocortisone treatment is a significant increase in the number of glutamate receptors in the forebrain of male Wistar rats. The increase in receptor number observed in this study is probably due to an increase in the synthesis of glutamate receptors and is associated with a marked reduction in the affinity of the glutamate receptors for glutamate. possible to demonstrate an receptor number or the For practical reasons, it was not effect of melatonin on either glutamate affinity of glutamate receptors for glutamate in rat forebrain membranes. In view of the neurotoxic effect of glutamate in the eNS, the functional significance of recently described glutamate receptors in the pineal gland was investigated. The results show that 10-4 M glutamate significantly inhibits the isoprenaline-stimulated synthesis of N-acetylserotonin and melatonin in organ culture when the pineal glands were pre-incubated with glutamate for 4 hours prior to stimulation with isoprenalin and when glutamate and isoprenaline were administered together in vitro. GABA, a glutamate metabolite could not mimic the decrease in isoprenalinestimulated melatonin, and it is likely that the observed effects were directly attributed to glutamate. Incubation of the pineal gland with 10-4 M glutamate in organ culture did not affect HIOMT activity in pineal homogenates, but significantly elevated both basal and isoprenaline-stimulated NAT activity. It was concluded that glutamate only inhibits melatonin synthesis in intact pineal glands and not in pineal homogenates. The present study has provided further support for an interaction between the pineal and the adrenal glands. There is an ever increasing likelihood that melatonin is an anti-stressogenic hormone and that the pineal gland may have a protective role to play in the pathology of stress-related diseases.

The pineal gland has become the subject of considerable investigation as it provides a productive experimental model for studying circadian rhythms and regulation of end organs. In the rat, the pineal gland provides a convenient model for investigating the noradrenergic receptor system and the effects of various drugs on this system. The effect of appetite suppressants on the rat pineal gland function is described. Appetite suppressants increase melatonin synthesis in organ cultures of rat pineal glands. This effect appears to be mediated by noradrenaline acting on β-adrenoceptors on the pinealocyte membrane. When β-adrenoceptors are blocked, the appetite suppressant-induced rise in melatonin synthesis is prevented. Depletion of noradrenaline in sympathetic nerve terminals also prevented the appetite suppressant-induced rise in melatonin synthesis. Activation of β-adrenoceptors is followed by a rise in N-acetyltransferase activity via a cyclic adenosine monophosphate second messenger system. The effect of appetite suppressants on the activity of liver tryptophan pyrrolase was also investigated. The activity of this enzyme is an important determinant of tryptophan availability to the brain and consequently of brain serotonin levels. The results show that appetite suppressants inhibit both holoenzyme and total enzyme activities of tryptophan pyrrolase. This finding suggests that appetite suppressants may act by inhibiting tryptophan pyrrolase activity thereby increasing brain serotonin, a phenomenon known to be associated with anorexia. There are two possible mechanisms by which appetite suppressants inhibit tryptophan pyrrolase activity. Firstly, these agents, being drugs of dependence, may increase liver NADPH concentrations which inhibit pyrrolase activity. Secondly, appetite suppressants may act on the pineal gland to stimulate melatonin synthesis. Melatonin inhibits pyrrolase activity in a dose-dependent manner. This inhibition will elevate plasma tryptophan levels which result in a rise in brain serotonin synthesis. The present study suggests a possible relationship between the pineal gland and appetite centres in the hypothalamus. Melatonin may have a direct effect on appetite centres since food restriction is associated with an increased melatonin binding in the hypothalamus. If this possible relationship can be extended, melatonin can open new possibilities for the control of food intake and consequently, of pathological obesity.