Serotonin metabolism in rat skin: characterization by liquid chromatography-mass spectrometry

We have recently uncovered the full expression of novel cutaneous serotoninergic and melatoninergic systems in the human and hamster skin. In this work, we have characterized serotonin metabolism in the rat skin using liquid chromatography-mass spectrometry and found that serotonin undergoes acetylation in the presence of acetyl coenzyme A. Inhibition of serotonin acetylation with Cole bisubstrate inhibitor shows that rat skin expresses both arylalkylamine and arylamine N-acetyltransferase activities. The serotonin degradation product-5-hydroxyindole acetic acid is also detected and pargyline (monoaminooxidase inhibitor) suppresses almost completely 5-hydroxyindole acetic acid accumulation. Together with previous data, the present study clearly demonstrates that biotransformation of serotonin in mammalian skin follows two alternate pathways. In the first pathway, serotonin is acetylated by arylalkylamine and arylamine N-acetyltransferases to generate the precursor of melatonin. Alternately, serotonin may undergo oxidative deamination by monoaminooxidase followed by enzymatic degradation by aldehyde dehydrogenase into 5-hydroxyindole acetic acid, which is presumably devoid of biological activity. Thus, the current methodological development of a liquid chromatography-mass spectrometry-based assay allows rapid resolution of the cutaneous metabolism of serotonin.     

The Effects of Caffeine and Caffeine-containing Beverages on the Disposition of Brain Serotonin in Rats

Caffeine and caffeine-containing beverages (instant coffee, black tea, green tea, or oolong tea) caused a significant decrease in serum tryptophan, and significant increases in brain tryptophan, serotonin, and 5-hydroxyindole acetic acid over those in rats fed a control diet. Adenosine supplementation partially counteracted the increase of brain serotonin caused by caffeine. These results are interpreted as indicating that caffeine-containing beverages may have some nutritional and behavioral effects.     

Serotonin catabolism in the central and enteric nervous systems of rats upon induction of serotonin syndrome

Serotonin, a well-known neurotransmitter in mammals, has been linked to a number of neurological and gastrointestinal disorders. One of these disorders, serotonin syndrome, is a potentially deadly condition caused by increased levels of serotonin in the extracellular space. Information on the neurochemical effects of serotonin syndrome on serotonin catabolism is lacking, particularly in relation to the enteric system of the gastrointestinal tract. Here the catabolism of serotonin is monitored in rats with pharmacologically induced serotonin syndrome, with the catabolites characterized using a specialized capillary electrophoresis system with laser-induced native fluorescence detection. Animals induced with serotonin syndrome demonstrate striking increases in the levels of serotonin and its metabolites. In the brain, levels of serotonin increased 2- to 3-fold in animals induced with serotonin syndrome. A major serotonin metabolite, 5-hydroxyindole acetic acid, increased 10- to 100-fold in experimental animals. Similar results were observed in the gastrointestinal tissues; in the small intestines, serotonin levels increased 4- to 5-fold. Concentrations of 5-hydroxyindole acetic acid increased 32- to 100-fold in the intestinal tissues of experimental animals. Serotonin sulfate showed surprisingly large increases, marking what may be the first time the compound has been reported in rat intestinal tissues.     

Determination of monoamines in brain nuclei by high performance liquid chromatography with electrochemical detection : Young vs. middle aged rats

A previously described high performance liquid chromatographic method for simultaneously measuring norepinephrine, dopamine, serotonin and 5-hydroxyindole acetic acid has been modified to permit analysis of the monoamines in microdissected brain nuclei from a single rat. Results of measurements in 12 nuclei of young and middle aged male rats are presented.     

Effect of sustained exercise on concentrations of plasma aromatic and branched-chain amino acids and brain amines

In both trained and untrained rats, exercise increased the plasma concentration ratio of aromatic amino acids to branched-chain amino acids which might favour entry of the aromatic amino acids into the brain. Exercise in trained rats did not change the brain concentration of 5-hydroxytryptamine but increased that of 5-hydroxyindole acetic acid. Exercise in the untrained rat increased the concentration of brain tryptophan and that of 5-hydroxytryptamine but that of 5-hydroxyindole acetic acid was unchanged. The increased concentration of 5-hydroxytryptamine in untrained rats might be involved in central fatigue.     

Dopamine and Serotonin Metabolism in Hepatic Encephalopathy

Patients with stupor or coma from fulminant hepatic failure were found to have high cerebrospinal fluid concentrations of homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA), metabolites of dopamine and serotonin respectively. Excessive amounts of their precursors—phenylalanine and tyrosine and free tryptophan—were found in the patients'' plasma. Methionine, which participates in dopamine degradation, was also much increased. Similar disturbances were found in patients suffering an acute exacerbation of chronic encephalopathy. These abnormalities would be consistent with other evidence of an increased turnover of serotonin and possibly dopamine in the brain during hepatic encephalopathy.     

Selective decrease in protein intake following brain serotonin depletion.

The effect of reduced brain serotonin concentration achieved with drugs or raphé lesions on the control of protein intake and energy intake by young rats was examined. All rats were provided an oppurtunity to select from 15% and 55% casein diets provided simultaneously in two food cups. Systemic parachlorphenylalanine, central 5, 7-dihydroxytryptamine or mid-brain raphé lesions reduced by 20–30% the amount of protein consumed from these two diets over a two week study period. Across these same groups total energy intake was not different from the control groups. This decrease in the proportion of dietary energy selected as protein by the treated self-selecting rats was associated with the reduction observed in brain serotonin and its major metabolite, 5-hydroxyindole acetic acid at the completion of the feeding period. These findings support previous work implicating brain serotonin metabolism in the selective control of protein intake.     

Increased tolerance of the dopamine- and serotoninergic systems during chronic administration of haloperidol and levomepromazine

In experiments on male albino rats single administration of haloperidol produced catalepsy, increase in dopamine turnover, enhancement of main dopamine metabolite homovanilinic acid in the forebrain. After single administration of the levomepromazine the cataleptogenic effect was accompanied by an enhanced 5-hydroxyindole acetic acid level, and no influence on the dopamine metabolism was observed. During chronic administration of haloperidol and levomepromazine their ability to induce catalepsy and to increase homovanilinic acid or 5-hydroxyindoleacetic acid concentration diminished. Thus, it appears that chronic administration of haloperidol reduces the sensitivity of dopamine receptors, and chronic administration of levomepromazine--reduces the sensitivity of dopamine and serotonin receptors in the brain.     

Improved Production of Human Immunoglobulin γ1 Chain Fc Polypeptides in Escherichia coli and Their Properties

Exposure to some xenobiotics (pentobarbital, 3-terf-butyl-4-methoxyphenol (BHA), chloretone (acetone chloroform), 1, l-bis-(p-chlorophenyl)-2,2,2-trichloroethane (DDT) and polychlorinated biphenyls (PCB)) for a 5 hr period increased the concentrations of brain serotonin and 5-hydroxyindole acetic acid (5HIAA). The decrease in the brain serotonin level elicited by /7-chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, was prevented by the concomitant administration of chloretone. The administration of both chloretone and pargyline (an inhibitor of monoamine oxidase) caused significant elevation of the brain 5HIAA level as compared with that in a pargyline control, however, the concentration of brain serotonin was not different between pargyline alone and chloretone plus pargyline. These results show that the increase in the brain serotonin level caused by chloretone is not due to acceleration of brain serotonin synthesis, but to retardation of the degradation of brain serotonin, and the increase in brain 5HIAA caused by chloretone may be due to the reduced removal of 5HIAA from the brain. Chloretone plus pargyline caused significant elevation of hypothalamus catecholamines, as compared to in the pargyline control, so the catecholamine turnover rates may be accelerated by the administration of chloretone.     

Acetaminophen inhibits liver trytophan-2,3-dioxygenase activity with a concomitant rise in brain serotonin levels and a reduction in urinary 5-hydroxyindole acetic acid

The effect of the analgesic agent, acetaminophen was determined on rat forebrain serotonin levels as well as hepatic tryptophan-2,3-dioxygenase (TDO) activity and urinary 5-hydroxyindole acetic acid (5-HIAA). The results show that acetaminophen administration (100mg/kg) over three hours does not affect the holoenzyme of tryptophan-2,3-dioxygenase but significantly inhibits the apoenzyme. This inhibition is accompanied by a concomitant rise in forebrain serotonin levels. This phenomenon is also accompanied by a reduction in urinary 5-HIAA levels. These results suggest that acetaminophen use is accompanied by changes in brain serotonin levels due to inhibition of hepatic tryptophan-2,3-dioxygenase activity. This in turn could explain the possible abuse potential of acetaminophen and its effects on mood at high doses.     

Long-term effects of 5,7-dihydroxytryptamine on brain monoamines

5,7-dihydroxytryptamine (75 and 150 μg) was injected intraventricularly to adult male rats; animals were killed at various times after the injection and brains were examined for changes in the concentration of tryptophan, serotonin, 5-hydroxyindole acetic acid, norepinephrine and dopamine. Brain 5-hydroxyindoleamines were markedly depleted at all time periods examined, even after the administration of a tryptophan load (50 mg/kg). A small but significant decline in brain norepinephrine but not dopamine was also noted after the administration of the dihydroxytryptamine.     

Increased tryptophan uptake into the brain in hyperammonemia

Hyperammonemia was provoked in rats by urease injection over three days. Tryptophan transport into the forebrain measured by the bolus injection technique was increased in hyperammonemic rats in comparison with pairfed controls. The concentration of the large neutral aminoacids, of tryptophan and of 5-hydroxyindole acetic acid were increased in the forebrain and brainstem. Probenecid administration led to a significantly higher accumulation of 5-hydroxyindole acetic acid in the forebrain of hyperammonemic rats. Since liver function was not impaired the data indicate that hyperammonemia in absence of hepatic insufficiency alters the carrier function for large neutral aminoacids at the blood brain barrier.     

Changes in the serotonergic system in the main olfactory bulb of rats unilaterally deprived from birth to adulthood

The serotonergic system plays a key role in the modulation of olfactory processing. The present study examined the plastic response of this centrifugal system after unilateral naris occlusion, analysing both serotonergic afferents and receptors in the main olfactory bulb. After 60 days of sensory deprivation, the serotonergic system exhibited adaptive changes. Olfactory deprivation caused a general increase in the number of fibres immunopositive for serotonin but not of those immunopositive for the serotonin transporter. HPLC data revealed an increase in serotonin levels but not in those of its major metabolite, 5-hydroxyindole acetic acid, resulting in a decrease in the 5-hydroxyindole acetic acid/serotonin ratio. These changes were observed not only in the deprived but also in the contralateral olfactory bulb. Double serotonin-tyrosine hydroxylase immunolabelling revealed that the glomerular regions of the deprived olfactory bulb with a high serotonergic fibre density showed a strong reduction in tyrosine hydroxylase. Finally, the serotonin(2A) receptor distribution density and the number of juxtaglomerular cells immunopositive for serotonin(2A) receptor remained unaltered after olfactory deprivation. Environmental stimulation modulated the serotonergic afferents to the olfactory bulb. Our results indicate the presence of a bilateral accumulation of serotonin in the serotonergic axon network, with no changes in serotonin(2A) receptor density after unilateral olfactory deprivation.     

Serotonin turnover in individual brain nuclei: evaluation of three methods using liquid chromatography with electrochemical detection

Liquid chromatography with electrochemical detection and brain microdissection techniques were used to evaluate three methods of studying serotonin turnover in 10 individual brain nuclei. The increase in serotonin (5-HT) and decline in 5-hydroxyindole acetic acid (5-HIAA) after administration of the monoamine oxidase inhibitor, pargyline, as well as the accumulation of 5-hydroxytryptophan (5-HTP) after the L-amino acid decarboxylase inhibitor, m-hydroxybenzylhydrazine, were measured. Serotonin accumulation and 5-HIAA decline could be detected in the n. caudatus, globus pallidus, cortical amygdala, n. interstitialis striae terminalis, n. preopticus medialis, and n. dorsomedialis. Only serotonin accumulation could be accurately assessed in the n. ventromedialis, n. arcuatus, and median eminence. The pattern of increase of serotonin after pargyline varied in different nuclei. There was a linear increase of serotonin over 90 minutes in the caudate, globus pallidus, and ventromedial nucleus and over 60 minutes in the n. preopticus medialis, and cortical amygdala. This contrasted with a maximal increase at 30 minutes in the other nuclei. However, 5-HIAA decline tended to be greatest after 30 minutes in most nuclei. Increases in 5-HTP concentrations after decarboxylase inhibition were not reliably detected in these areas. These results indicate that two nonsteady state methods may be used to evaluate changes in serotonin turnover in selected individual, nonpooled hypothalamic and forebrain nuclei.     

Effect of intravenous 5-hydroxytryptophan on hypothalamic concentration of norepinephrine, dopamine, serotonin and hydroxyindole acetic acid in the fetal lamb

To investigate the neurochemical mechanism of the response of growth hormone to 5-hydroxytryptophan (5-HTP), we administered 5-HTP (20 mg/kg) to 10 ovine fetuses (110 or 130 days old; term gestation 147 days). Ninety minutes after 5-HTP administration, and following increases in plasma growth hormone concentrations, the fetus was delivered by hysterotomy. After local anesthesia of the fetus and sacrifice by cervical spinal cord transection the hypothalamus rapidly dissected, and stored at -80 degrees C for later analysis of norepinephrine, dopamine, serotonin and hydroxyindole acetic acid. Compared to the administration of saline, 5-HTP caused a significant increase in the hypothalamic content of serotonin, and norepinephrine, at both gestational ages. 5-hydroxyindole acetic acid increased significantly only in the older fetuses. These results indicate that serotonin may not be the only neurotransmitter active in the growth hormone response to 5-HTP.     

Pyruvate as a fluorescence quencher: a new spectroscopic assay for pyruvate reactions

Optimal conditions for the extraction from brain tissue and the simultaneous quantification of catechol and indole derivatives were determined after a systematic degradation study in water and perchloric acid. The roles of three parameters, namely temperature, presence of antioxidant agents, and time, were considered. Adrenaline, noradrenaline, dopamine, homovanillic acid, 5-hydroxytryptophan, 5-hydroxyindole acetic acid, serotonin, and epinephrine were separated by HPLC and detected electrochemically. The results indicated a great instability of the indole derivatives at an ambient temperature, in an acid medium, and in the absence of a protective agent. Therefore, when perchloric acid has to be used for deproteinization, the lowest concentration (0.1 M) is preferable. The samples have to be kept on ice, in darkness, and protected by ascorbic acid and sodium ethylenediamine tetracetate.     

Plasma transport of 5-hydroxytryptamine-3H. II. Identification of the carrier protein of 5-hydroxyindoleacetic acid]

In the first note, we have demonstrated a 5-hydroxyindole acetic acid binding to plasma proteins. By gel filtration on Sephadex G 200 and polyacrylamide gel electrophoresis, the 5-hydroxyindole acetic acid carrier protein was identified to serum-albumin.     

Influences of cyclooxygenase inhibitors on the cataleptic behavior induced by haloperidol in mice.

Haloperidol administered intraperitoneally, and prostaglandin F2 alpha (PGF2 alpha) and PGE2 intraventricularly induced dose-dependent cataleptic behavior in mice. The cataleptic behavior induced by haloperidol was inhibited dose-dependently by oral pretreatment with aspirin and indomethacin, inhibitors of PGs synthetase. Striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole 3 acetic acid (5-HIAA) were elevated by haloperidol, although dopamine (DA) and 5-hydroxytryptamine (5-HT) levels did not change. The increase of DOPAC level in striatum induced by haloperidol was significantly suppressed by aspirin, but not in brain stem. The alteration of DOPAC level by aspirin correlated with the behavioral response. These results suggest that central prostaglandin synthesis may participate in the development of cataleptic behavior, which might also involve alteration of brain catecholaminergic activity.     

Caffeine injection raises brain tryptophan level,but does not stimulate the rate of serotonin synthesis in rat brain

Acute caffeine injection (100 mg/kg) elevates brain levels of tryptophan (TRP), serotonin (5HT), and 5-hydroxyindoleacetic acid (5HIAA). Experiments were performed to determine if the increases in 5HT and 5HIAA result from a stimulation of the rate of 5HT synthesis. Both the rate of 5-hydroxytryptophan (5HTP) accumulation following NSD-1015 injection, and the rate of ³H-5-hydroxyindole synthesis from ³H-tryptophan were measured $\text{in vivo}$ following caffeine administration and found to be normal. Tryptophan hydroxylase activity, as measured $\text{in vitro}$ in brain homogenates, was also unaffected by caffeine. The results suggest that the elevations in brain 5HT and 5HIAA levels produced by caffeine do $\text{not}$ reflect enhanced 5HT synthesis, despite significant elevations in brain TRP level. Some other mechanism(s) must therefore be responsible for these elevations in brain 5-hydroxyindole levels.     

Serotonin catabolism and the formation and fate of 5-hydroxyindole thiazolidine carboxylic acid

Serotonin (5-HT) functions as a neurotransmitter and neuromodulator in both the central and enteric nervous systems of mammals. The dynamic degradation of 5-HT metabolites in 5-HT-containing nervous system tissues is monitored by capillary electrophoresis with wavelength-resolved laser-induced native fluorescence detection in an effort to investigate known and novel 5-HT catabolic pathways. Tissue samples from wild type mice, genetically altered mice, Long Evans rats, and cultured differentiated rat pheochromocytoma PC-12 cells, are analyzed before and after incubation with excess 5-HT. From these experiments, several new compounds are detected. One metabolite, identified as 5-hydroxyindole thiazoladine carboxylic acid (5-HITCA), has been selected for further study. In 5-HT-incubated central and enteric nervous system tissue samples and differentiated PC-12 cells, 5-HITCA forms at levels equivalent to 5-hydroxyindole acetic acid, via a condensation reaction between L-cysteine and 5-hydroxyindole acetaldehyde. In the enteric nervous system, 5-HITCA is detected without the addition of 5-HT. The levels of L-cysteine and homocysteine in rat brain mitochondria are measured between 80 and 140 microm and 1.9 and 3.4 microm, respectively, demonstrating that 5-HITCA can be formed using available, free L-cysteine in these tissues. The lack of significant accumulation of 5-HITCA in the central and enteric nervous systems, along with data showing the degradation of 5-HITCA into 5-hydroxyindole acetaldehyde, suggests that an equilibrium coupled to the enzyme, aldehyde dehydrogenase type 2, prevents the accumulation of 5-HITCA. Even so, the formation of 5-HITCA represents a catabolic pathway of 5-HT that can affect the levels of 5-HT-derived compounds in the body.