Factors affecting tryptophan hydroxylase in the brain and pineal gland

Abstract

The aim of this investigation was to study factors which affect tryptophan hydroxylase in the brain and pineal gland. My studies have shown that the expression of tryptophan hydroxylase activity is markedly suppressed in the presence of particulate fractions of rat midbrain tissue. Evidence that enzyme(s) which inactivate tetrahydrobio-pterin are present in these particulate fractions, is presented. Tetrahydrobiopterin incubated at 37ºC with either homogenates or purified membrane fractions from rate midbrain rapidly loses its ability to act as a co factor for tryptophan hydroxylation. Anaerobic conditions greatly diminish this loss in cofactor activity suggesting the process involves an oxidation of the pteridine. KCN fails to inhibit the inactivation. The incubation of tetrahydrobiopterin with partially purified membrane fraction from rat midbrain is also accompanied by marked alterations in its spectral characteristics. Both the loss in cofactor activity and the spectral changes are prevented by prior boiling of the particulate fractions. is hypothesised that the apparent suppression of tryptophan hydroxylase activity in homogenate fractions of rat midbrain tissue is mediated by those factors present in the membrane fraction which inactivate tetra-hydrobiopterin. Some relevant properties of tryptophan hydroxylase were studied. The enzyme was found to lose activity rapidly even at 0°C. Both tryptophan and tetrahydrobiopterin slightly but significantly reduced the level to which the enzyme decayed at 37°C. Thiol containing compounds were shown to substantially protect pineal tryptophan hydroxylase from inactivation at 0°C but provided little protection at higher temperatures. The inactivation process appeared to be independent of oxygen The activity of the enzyme, lost after ageing at 0°C, could be recovered by incubation with dithiothreitol under anaerobic conditions. Fresh enzyme, or enzyme inactivated at J7°C could not be activated by this process. Tryptophan hydroxylase in the pineal gland of the rat was found to undergo a diurnal rhythm in activity with an elevated activity at night. The rhythm was abolished in constant light. Cycloheximide (15 mg/kg, i.p.)? administered both at night and during the day, caused a rapid, decay in activity suggesting that tryptophan hydroxylase was subject to a rapid turnover in vivo. A rapid decay in tryptophan hydroxylase activity was also observed in isolated pineal glands cultured in the presence of either cycloheximide or puromycin. The primary site of control appears to be at the level of translation since Actinomycin D had little or no effect. The mechanism by which the increase in tryptophan hydroxylase activity occurs, at night was investigated. Isoproterenol administered during the day caused a significant increase in enzyme activity in vivo suggesting that the release of noradrenaline from sympathetic nerves on to p-adrenergic receptors may cause the night time elevation in tryptophan hydroxylase activity. This hypothesis is further supported by the fact that a marked increase in enzyme activity was also observed in isolated glands cultured in the presence of noradrenaline or isoproterenol (in an atmosphere of 95 percent C>2 + 5 percent C02). The fact that dBcAMP also significantly elevates enzyme activity suggests the process may be mediated by cAMP. The increases are unlikely to be secondary to an accumulation of melatonin in the pineal gland, since the administration of melatonin (5 mg/kg) during the day failed to alter enzyme activity. Studies have shown that the maintainence of tryptophan hydroxylase activity in pineal glands in culture is markedly affected by the concentration of 02 in the gaseous atmosphere used. A rapid decline in enzyme activity occurs in glands cultured in an atmosphere of air instead of 95 percent 02. In addition tryptophan hydroxylase activity becomes unresponsive to either noradrenaline or dBcAMP in glands cultured in air. A re-examination of the action of p-chlorophenylalanine (PCPA) on pineal tryptophan hydroxylase revealed that an irreversible inactivation occurred within 6 h (2% of initial activity) followed by a recovery within 24 hours. The rapid turnover of the enzyme is the probable reason for the failure of previous studies to observe an irreversible inhibition of this enzyme by PCPA, in vivo. A commercially available salt of serotonin 5HT oxalate, was found to contain a potent inhibitor of both pineal and midbrain tryptophan hydroxylases. Although the inhibitor has not yet been identified its actions can be readily dissociated from those of 5HT or oxalic acid.