Conversion of 5-hydroxytryptophan into serotonin by tryptophan decarboxylase in plants, Escherichia coli, and yeast

The L-tryptophan decarboxylase (TDC) gene of rice was heterologously expressed in various organisms. Transgenic rice overexpressing TDC showed accumulation of serotonin upon 5-hydroxytryptophan treatment, which was consistent with the in vitro 5-hydroxytryptophan decarboxylase enzyme activity of purified recombinant rice TDC in a pyridoxal phosphate-dependent manner. Recombinant yeast harboring TDC produced serotonin at the expense of the endogenous 5-hydroxytryptophan levels.     

The effect of 5-hydroxytryptophan on shivering thermogenesis]

Studies carried out on ground squirrels showed active warming of these animals during arousal from hibernation to be closely connected with intensification of the shivering thermogenesis. The intraabdominal administration of 5-oxytryptophane, a precursor of serotonin clearly suppressed the shivering thermogenesis in arising ground squirrels and slowed down their warming.     

New degradative routes of 5-hydroxytryptophan and serotonin by intestinal tryptophan 2,3-dioxygenase

A highly purified preparation of tryptophan 2,3-dioxygenase from rabbit intestine was found to catalyze the oxygenative ring cleavage of 5-hydroxytryptophan and serotonin. The products of these enzymic reactions were susceptible to the action of formamidase, as a consequence of which 5-hydroxykynurenine and 5-hydroxykynurenamine were isolated from the reaction mixtures and identified. The initial products were presumed, therefore, to be 5-hydroxyformylkynurenine and 5-hydroxyformylkynurenamine, respectively. Several lines of evidence indicated that the cleavage of tryptophan, 5-hydroxytryptophan and serotonin occurred by the action of a single protein, namely intestinal tryptophan 2,3-dioxygenase.     

The effect of repeated tryptophan administration on body weight, food intake, brain lipid peroxidation and serotonin immunoreactivity in mice

Tryptophan as a circulating precursor of serotonin (5-HT) may suppress food intake and body weight. Tryptophan administration can enhance the generation of reactive oxygen species (ROS) by inducing oxidative pathway in vivo and in vitro. We have examined the effect of repeated tryptophan administration on food consumption, body weight, brain lipid peroxidation and 5-HT immunoreactivity. Tryptophan was given at the dose of 100 mg/kg/24 hr in 0.2 ml saline solution i.p. for 7 days to mice. Control mice received 0.9% NaCL solution at the same manner and volume. Body weights were recorded at the beginning and end of the experiments. Thiobarbituric acid reactive substance (TBARS), the last product of lipid peroxidation, was measured spectrophotometrically. Brain 5-HT levels were determined by the immunohistochemical method. Our findings indicate that the tryptophan suppresses food intake significantly in mice. Body weight decreased and brain TBARS levels increased significantly by repeated tryptophan treatment. Immunohistochemical detection showed that 5-HT levels increased by tryptophan administration. There is a link between increased 5-HT level and oxidative stress by tryptophan administration on brain tissue. Tryptophan at repeated doses should be exercised carefully in clinical practice.     

Effects of 5-hydroxytryptophan on serotonin in nerve endings

—Preparations of synaptosomes (P₂) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of 5-hydroxytryptophan (5-HTP) on (a) the levels of serotonin (5-HT) in nerve endings and (b) the release of 5-HT from nerve endings. The levels of 5-HT were significantly higher (3.21 × 0.35 nmol/g original tissue weight) in the P₂ fraction isolated from the telencephalon of pigeons given intramuscular injections of 50mg/kg of d ,l -5-HTP in comparison to control values (1.42 ± 0.07). A similar twofold increase was observed with the P₂ fraction isolated from the diencephalon plus optic lobes. In addition, the levels of 5-HTP and 5-hydroxyindoleacetic acid also increased significantly in these P₂ fractions isolated from pigeons given d ,l -5-HTP injections in comparison to values obtained for pigeons given saline injections. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [³H]5-HT indicated that 0.10 mil l -5-HTP increased the release of [³H]5-HT twofold over control values. A concentration as low as 0.001 mm l -5-HTP was tested on the P₂ fraction from the telencephalon of the pigeon and was found to significantly increase the release of [³H]5-HT over control values. This effect by l -5-HTP was blocked if a decarboxylase inhibitor was added to the medium. l -5-HTP at a concentration of 1.5 mm had no apparent effect on the release of [³H]norepinephrine or [³H]dopamine from synaptosomes prepared from the telencephalon of the rat or pigeon. The results are discussed in terms of the role of serotonin in producing certain types of behavioral depressions exhibited by pigeons and rats given injections of 5-HTP.     

Effects ofp-chlorophenylalanine on the metabolism of serotonin from 5-hydroxytryptophan

The effects ofD,L--chlorophenylalanine methyl ester (PCPA-methyl ester) and two of its metabolites, 2-(-chlorophenyl)-ethylamine (PCPEA) and -chlorophenylacetic acid (PCPAA), on the metabolism of serotonin (5-HT) fromD,L-5-hydroxytryptophan (5-HTP) ware studied in vitro and in vivo using the telencephalon and brainstem of the rat. For in vivo studies and some in vitro experiments, rats were injected with either 100 mg/kg PCPA-methyl ester or saline alone on days 1, 2, and 3, and were killed on day 15. When the in vivo metabolism of 5-HT was to be studied, the saline group and the PCPA group of animals were injected with 75 g/kg [³H]D,L-5-HTP 20 min before sacrificing. With respect to the values found for the saline-injected animals, the specific activity (S.A.; dpm/nmol) of 5-HIAA was significantly greater in the telencephanol and brainstem of the animals injected with PCPA-methyl ester. The S.A. of 5-HTP was the same in both groups; the S.A. of 5-HT was lower in the telencephalon of the PCPA group than in the saline group; in the brainstem, there was no difference. In both the saline- and PCPA-injected animals, the S.A. of 5-HIAA was greater than the S.A. of 5-HT. There was no difference between the saline- and PCPA-injected animals with regard to: (1)L-5-HTP decarboxylase activity; (2)L-5-HTP-induced release of [³H]5-HT in vitro from crude nerve ending fractions (P₂); or (3) in vitro uptake of [³H]D,L-5-HTP and its conversion to [³H]5-HT using the P₂ fraction. In vitro studies demonstrated that the PCPEA could directly cause a large increase in the release of [³H]5-HT from the P₂ fraction, whereas PCPA and PCPAA had little or no apparent effect. The data were interpreted to suggest that in the telencephalon of the animals treated with PCPA-methyl ester, there was a higher turnover of 5-HT than was found in the saline-treated group.     

Neurochemical correlates of behaviour--content of tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, tyrosine, dopamine and norepinephrine in four brain parts of the pigeon during behavioural depression following an injection of tryptophan.

Abstract— Pigeons working on a multiple lixed-ratio 50, fixed interval 10 schedule of food reinforcement were injected with l -tryptophan (300mg/kg; I.M.) and killed at various times before, during and after the period of behavioural depression following the administration of this amino acid (0, 25, 50, 90, 170 and 230 min). The levels of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tyrosine, dopamine and norepinephrine were concurrently measured in 4 specific areas of the brain (telencephalon, diencephalon plus mesencephalon, pons plus medulla-oblongata and cerebellum). The course of the increases in the level of 5-hydroxytryptamine in the telencephalon, and subsequent return to pre-injection levels, was temporally related to the onset of the decreased responding and gradual return to normal rates of responding. Changes in dopamine and norepinephrine were not correlated with the onset of and recovery from the decreased response rates. The data in this paper are discussed in terms of (a) the previously reported work with 5-hydroxytryptophan and (b) the importance of the telencephalic serotonergic system in certain types of behavioural depression.     

Isotopic microassay of histamine, histidine, histidine decarboxylase and histamine methyltransferase in brain tissue

Abstract— Microassays are described for histamine, histidine, and the activities of the enzymes histidine decarboxylase (EC 4.1.1.22) and histamine niethyltransferase (EC 2.1.1.8) in brain tissue. The enzymic-isotopic microassay for histamine is based on the methylation of tissue histamine by added histamine methyl-transferase and [¹⁴C]- or [³H]-labelled S-adenosyl-l -methionine. In a double-isotopic form of the assay, a tracer of [³H]histamine is employed along with [¹⁴C]S-adenosyl-l -methionine, and the ratio [¹⁴C]:[³H] reflects the amount of histamine in the sample. Because the methylation of histamine is uniform in brain samples studied, a single isotopic assay with [³H]S-adenosyl-l -methionine as the methyl donor is possible and increases sensitivity, so that 10 pg of tissue histamine can be estimated reliably. The assay for histidine involves decarboxylation of histidine by a bacterial histidine decarboxylase and measurement of the histamine formed by the enzymicisotopic procedure. In the histidine decarboxylase assay, histamine synthesized from added histidine is measured. The assay for histamine methyltransferase involves measuring the formation of [¹⁴C]methylhistamine with [¹⁴C]S-adenosyl-l -methionine serving as the methyl donor.     

Comparison of the cardiovascular responses to intracerebroventricular administration of tryptamine, 5-hydroxytryptamine, tryptophan and 5-hydroxytryptophan in rats

General characteristics of the cardiovascular responses to intracerebroventricular (i.c.v.) injection of tryptamine, 5-hydroxytryptamine (5-HT), tryptophan and 5-hydroxytryptophan (5-HTP) were compared. Relatively small doses of tryptamine and 5-HT (0.005-0.1 microM) produced considerable, long-lasting and dose-dependent pressor effects, which sometimes were followed by prolonged depressor effects. Tryptophan (0.02-0.5 microM) and 5-HTP (0.02-0.2 microM) caused variable and usually slight, but long-lasting, vascular responses or no vascular response A large dose of tryptamine (0.5 microM) evoked variable vascular effects, while the same dose of 5-HT and 5-HTP evoked marked and prolonged depressor effects. The vascular responses to the drugs were accompanied by variable changes in heart rate. Tryptamine, 5-HT and 5-HTP, in the majority of rats, produced a bradycardia. The present study provides evidence that the cardiovascular response to i.c.v. administration of tryptamine is similar to that of 5-HT, supporting the idea that tryptamine, in addition to 5-HT, participates in the central physiological regulation of the rat cardiovascular system. The role of tryptophan and 5-HTP by themselves in this regulation, if any is of secondary importance.     

Uptake of serotonin, 5-hydroxytryptophan and tryptophan by giant serotonin-containing neurones and other neurones in the central nervous system of the snail (Helix pomatia)

Summary Following exposure to tritiated 5-HTP, silver grains were observed over the perikarya of the GSCs (Giant serotonin cells) of Helix pomatia and other known serotonin-containing neurones in light and electron microscope autoradiograms. There was no indication that the 5-HTP was taken up by nerve endings or by non-nervous structures. The distribution of radioactivity was completely different in autoradiograms of tissue exposed to tritiated serotonin. Silver grains, often in very high concentrations, were observed only over certain fine axon branches and processes thought to be nerve endings. Electron microscope autoradiography showed that these processes contained small dense-cored vesicles, morphologically identical to those thought to sequester serotonin in the perikarya of the GSCs. The accumulation of tritiated tryptophan was less specific; all the neurone perikarya showed an accumulation of radioactivity after exposure to this substance.We are grateful to Professor J. F. Lamb for the use of the Scintillation Spectrometer.