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Both hydroxy- and methoxyindoles modify basal temperature in the rat.

The various pineal gland tryptophan metabolites were administered to male rats intraperitoneally (100 micrograms/kg) and rectal temperatures were recorded. Of the compounds tested, hydroxytryptophan, N-acetylserotonin, hydroxytryptophol, and their corresponding methoxyindoles all caused a marked hypothermia, indicating that several indolic products may be involved in thermoregulation. Although the brain penetration of indoles is poor, a central site of action would be most likely, although peripheral actions cannot be excluded. The mechanism of induction of hypothermia may involve peptides, the pituitary-thyroid axis, the adrenal gland, or a combination of these. These results may suggest that the pineal gland integrates environmental cues to act in concert with physiological thermostats in the fine tuning of thermoregulation.

Animals↗

Activity of melatonin and other pineal indoles on the in vitro synthesis of cortisol, cortisone, and adrenal androgens.

The in vitro effects of 13 indole compounds on the synthesis of glucocorticoids and of adrenal androgens in sheep adrenal glands has been studied from 11-deoxycortisol as a precursor. This work demonstrates the activating effect of some indole compounds on 11 beta-hydroxylase and 17,20-desmolase and the inhibitory effect of most of them on 11 beta-hydroxysteroid dehydrogenase. Three categories could be distinguished: 1) compounds without any effect (5-hydroxytryptophan, 5-hydroxytryptamine); 2) compounds moderately increasing (10-30% as compared with controls) cortisol yields (tryptamine, melatonin, 6-hydroxymelatonin, 5-methoxytryptophol, indomethacin); and 3) compounds markedly increasing (80-100%) cortisol yields (5-methoxyindole acetic acid, 5-hydroxyindole acetic acid, 2-methylindole, 5-hydroxytryptophol, N-acetyl-5-hydroxytryptamine). In fact, since most of the studied indoles reduced 11 beta-hydroxysteroid dehydrogenase activity, the actual activation of cortisol synthesis was four to five times less. Lastly, all the studied compounds, but melatonin, increased the activity of 17,20 desmolase as seen from 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione yields. The possible in vivo effects of the indoles for therapeutic use needs further studying.

Adrenal Glands↗

Antiproliferative effect of pineal indoles on cultured tumor cell lines.

The in vitro antiproliferative action of pineal indoles on several tumor cell lines including melanoma (B16), sarcoma (S180), macrophage-like cell line (PU5), fibroblasts (3T3), and choriocarcinoma (JAr) was examined by measuring the incorporation of 3H-thymidine by the tumor cells, and, in the case of melanoma cells, by also measuring the incorporation of 3H-leucine and 3H-uridine. Uptake of crystal violet was used to assess the viability of the tumor cells. The order of inhibitory potency of the indoles was found to be methoxytryptamine > melatonin, methoxytryptophol, hydroxytryptophol, and methoxyindoleacetic acid > serotonin and hydroxyindoleacetic acid. The possibility of an adverse effect of the indoles on the viability of normal cells was also investigated by employing a primary culture of rat hepatocytes. The release of glutamate-oxaloacetate transaminase by hepatocytes was not affected by the indoles, although the release of glutamate-pyruvate transaminase was increased to a small extent and the uptake of crystal violet was slightly inhibited.

3T3 Cells↗

Hydroxyindole-O-methyltransferase activity in ocular and brain structures of rabbit and hen.

Relative activities of hydroxyindole-O-methyltransferase (HIOMT) of some brain and ocular structures of the rabbit and hen were analyzed using different 5-hydroxyindoles, i.e., N-acetylserotonin (NAS), 5-hydroxytryptophol (HTOL), 5-hydroxytryptophan (HTP), 5-hydroxytryptamine (HT), and 5-hydroxy-3-indoleacetic acid (HIAA), as enzyme substrates. Pineal glands of both species, as well as hen retina, are capable of producing, to varying degrees, melatonin, 5-methoxytryptophol, and 5-methoxytryptamine. Hen choroid and iris-ciliary body O-methylated NAS and HTOL, whereas rabbit choroid and, to a much lesser extent, hypothalamus and cerebral cortex all O-methylated only NAS. No measurable HIOMT activity was found in hen brain. NAS was a preferred substrate for HIOMT in the hen tissues, whereas in the rabbit pineal gland NAS and HTOL were equally good substrates for HIOMT. Other tested 5-hydroxyindoles, i.e., HTP, HT, and HIAA, were poor methyl acceptors. Of the tissues examined, the highest HIOMT activity was found in the hen pineal gland, followed by the rabbit pineal gland and hen retina. No significant differences between day and nighttime enzyme activities were observed in the pineal gland and retina of either species. The data suggest that in vertebrates some nervous and ocular tissues possess the potential to produce 5-methoxyindole compounds; however, the HIOMT-catalyzed process shows remarkable substrate-, tissue- and species-dependent variations.

Acetylserotonin O-Methyltransferase↗

UDP glucuronosyltransferase (UGT) 1A6 pharmacogenetics: II. Functional impact of the three most common nonsynonymous UGT1A6 polymorphisms (S7A, T181A, and R184S).

The objective of this study was to use recombinant enzymes and human liver microsomes (HLMs) to comprehensively evaluate the functional impact of the three most common nonsynonymous polymorphisms (S7A, T181A, and R184S) identified in the human UDP glucuronosyltransferase (UGT) 1A6 gene. In addition to the known allozymes, other possible amino acid variants were expressed in human embryonic kidney (HEK)293 cells to enable structure-function analysis. Initial studies using different substrates (serotonin, 5-hydroxytryptophol, 4-nitrophenol, acetaminophen, and valproic acid) showed similar results with 2-fold higher glucuronidation by UGT1A6(*)2 (S7A/T181A/R184S) compared with UGT1A6(*)1 (reference), and intermediate activities for other variants. Enzyme kinetic analyses with the UGT1A6-specific substrate (serotonin) showed 50% lower K(m) values for all R184S variants and 2-fold higher V(max) values for both S7A/T181A variants compared with UGT1A6(*)1. Furthermore, intrinsic clearance (V(max)/K(m)) values were highest for the UGT1A6(*)2 allozyme (2.3-fold over UGT1A6(*)1), resulting from additive effects of higher enzyme affinity and activity. As expected, K(m) values of (*)1/(*)1 genotyped HLMs (5.4 +/- 0.2 mM) were similar to recombinant UGT1A6(*)1 (5.8 +/- 0.6 mM). Conversely, (*)2/(*)2 HLMs showed higher K(m) values (7.0 +/- 0.3 mM) rather than the lower K(m) values displayed by recombinant UGT1A6(*)2 (3.6 +/- 0.3 mM), suggesting that this allozyme may display different enzyme kinetic behavior in HLMs compared with HEK293 cells. At best, these polymorphisms were predicted to account for 15 to 20% of the observed 13-fold variability in glucuronidation of UGT1A6 substrates by HLMs, indicating that there are likely other genetic or environmental factors responsible for the majority of this variation.

Adolescent↗

Binding sites for melatonin in bovine pineal gland.

High-affinity binding of melatonin to crude membrane preparations of bovine pineal gland was examined by a rapid filtration procedure through Whatman GFB paper. Maximal melatonin binding was attained in 60 min at 37 degrees C, in 2 h at 25 degrees C and in 5 h at 0 degree C; at 25 and 37 degrees C it was 36 and 42% of that at 0 degree C. Specific binding was thermolabile and decreased following incubation with trypsin; it was also pH dependent, the maximum being observed at physiological pH. Melatonin binding was inhibited by the addition of monovalent or divalent ions to the incubation buffer. Subcellular fractionation studies indicated that 39 and 50% of binding was located in the pellets at 900 and 27,000 g whereas 11% was detected in the microsomal pellet. Scatchard analysis revealed a single population of binding sites with Kd = (7.0 +/- 1.5) 10(-7) M (mean +/- SEM, n = 4); binding site concentration ranged from 185 to 356 fmol/mg of protein. When various melatonin analogues were tested for their ability to inhibit (3H)-melatonin binding, the following Ki values (10(-7) M), were obtained: N-acetyl-serotonin 120, serotonin 130, 2-methyl indole 154, 5-hydroxytryptophol 218, 5-methoxytryptamine 266, 5-methoxytryptophol 660, tryptamine 1,740, 5-hydroxyindole acetic acid 3,455, 5-methoxyindole acetic acid 12,690, 5-hydroxytryptophan 13,600, and 6-hydroxymelatonin 55,550. Those results suggest that melatonin receptors may be present in the pineal gland.

Animals↗

Phagocytic activity of cultured retinal pigment epithelial cells from chick embryo: inhibition by melatonin and cyclic AMP, and its reversal by taurine and cyclic GMP.

Cultured chick retinal pigment epithelial cells phagocytosed polystyrene latex particles. The phagocytosis was inhibited very specifically by melatonin, which attained 50% inhibition at about 10(-16) M. Other indole compounds such as 5-methoxytryptophol, 5-hydroxytryptophol, 6-hydroxymelatonin, N-acetylserotonin, 5-methoxytryptamine and serotonin were also inhibitory although their effects were less than 1/10,000 that of melatonin. Possible retinal neurotransmitters, acetylcholine, gamma-aminobutyric acid, glycine, dopamine, aspartic acid and glutamic acid, had no or only a minimum inhibitory effect, and was also the case for prostaglandin D2, E2, F2 alpha, and I2. Taurine was not inhibitory at all. Among nucleotides, cyclic AMP specifically inhibited phagocytosis, giving 50% inhibition at about 10(-11) M. Melatonin inhibition was increased by copresence of isobutylmethylxanthine. Inhibition by either melatonin or cyclic AMP was reversed by dibutyryl cyclic GMP. The reversal was observed also with compounds which were expected to increase intracellular cyclic GMP. Prostaglandin D2 reversed inhibition in both cases, but its effect was incomplete and per se it had an inhibitory effect. Melatonin derivatives reversed inhibition by melatonin alone but not inhibition by cyclic AMP. Taurine efficiently reversed both kinds of inhibition. Other possible neurotransmitters were ineffective. Taurine was thus the most effective of these compounds. We suggest the following hypothetic control mechanism of phagocytic activity of the pigment epithelial cells: melatonin and cyclic AMP are intercellular and intracellular signals, respectively, of stopping phagocytosis, while taurine and cyclic GMP are intercellular and intracellular signals, respectively of cancelling this stop signal. Phagocytic activity of chick retinal pigment epithelial cells might be regulated by the concentration ratio of melatonin to taurine in the interphotoreceptor space.

Animals↗

Inhibition of the in vitro pituitary response to luteinizing hormone-releasing hormone by melatonin, serotonin, and 5-methoxytryptamine.

The effects of pineal indole compounds on the response of the neonatal rat anterior pituitary gland to LH-releasing hormone (LHRH) were studied in organ culture. After 24 h of culture under control conditions, pituitary glands from 5-day-old female rats were routinely incubated for an additional 24 h with the test compounds. Medium LH content was determined by double antibody radioimmunoassay. LHRH (10(-9) M) induced a 10-fold increase in LH levels over control values. Melatonin at a concentration of 10(-9) M significantly reduced the LHRH-stimulated release of LH; maximal suppression to 14% was attained with 10(-8) M melatonin. Similarly, 10(-8) M LHRH caused a 26-fold elevation in medium LH which was suppressed to 62, 55, and 47% by 10(-9), 10(-8), and 10(-7) M melatonin, respectively. In short-term experiments, inhibition was evident within 30 min of treatment. A developmental study of the effect of melatonin on LH release revealed significant inhibition with pituitary glands from rats 2, 5, and 10 days of age but not with glands from animals 21 and 30 days of age. Other pineal indoles were tested for their effects on the LH response to a single dose of LHRH (3 x 10(-10) M). Serotonin at a concentration of 10(-9) M significantly suppressed LH release; maximal reduction to 37-53% occurred with 10(-8) to 10(-6) M serotonin. 5-Methoxytryptamine also produced an inhibition which was significant only at 10(-6) M, the highest concentration tested. N-Acetylserotonin, 5-hydroxyindoleacetic acid, 5-methoxyindoleacetic acid, 5-hydroxytryptophol, and 5-methoxytryptophol showed no consistent inhibitory activity at doses up to 10(-7) M. These findings indicate that melatonin, serotonin, and 5-methoxytryptamine can act directly on the neonatal pituitary gland to suppress LHRH-induced release of LH.

5-Methoxytryptamine↗

Influence of exogenous melatonin on melatonin secretion and the neuroendocrine reproductive axis of intact male rats during sexual maturation.

The influence of daily sc administration of melatonin (5-100 micrograms/day) on sexual development of prepubertal and pubertal male rats was studied in vivo. Adult animals were also studied. When melatonin was injected daily into young animals starting at day 20 of age, dose-dependent reductions in plasma testosterone, testis and seminal vesicles weights, plasma FSH and LH levels, and pituitary GnRH receptor number were observed at day 40 or 45 of age when animals were killed. In contrast, prepubertal animals (5-20 days old) showed no significant responses to similar treatment with melatonin, whereas in adult animals (70-90 days old), melatonin elicited only a small decrease in plasma testosterone concentration. Melatonin analogs such as N-acetyl-serotonin and 5-hydroxytryptophol administered daily from 20-45 days of age did not produce any effect. Chronic melatonin administration from 20-50 days of life did not alter the occurrence of the nocturnal rise of circulating plasma melatonin, but did enhance its amplitude. Our results demonstrate that exogenous melatonin can inhibit or delay sexual maturation in the male rat if administered between 20 and 40 days of age and suggest that this inhibitory action is exerted at the hypothalamic and/or pituitary level.

Animals↗

Localization and release of 5-hydroxytryptamine in the crayfish eyestalk

The content and regional distribution of 5-hydroxytryptamine (5-HT) in the crayfish eyestalk was determined by high-performance liquid chromatography. Levels of the 5-HT precursors l-tryptophan (L-TRP) and 5-hydroxytryptophan (5-OH-TRP), and of three metabolites, 5-hydroxytryptophol (5-HTPH), N-acetylserotonin (NA-5-HT) and 5-hydroxy-indole-3-acetic acid (5-HIAA), were also determined. The total content of 5-HT in the eyestalk was 95.4+/-49.3 pg mg-1 wet mass (mean +/- s.d., N=55) while the specific content was 9.6+/-4.9 fmol microg-1 protein (mean +/- s.d. N=5). 5-HT was present in all four ganglia of the eyestalk. The highest proportion was found in the medulla terminalis (40.2 %) and the lowest in the retina lamina ganglionaris (9.9 %), which also had the lowest specific content. Conversely, the highest specific content of L-TRP was in the retina lamina ganglionaris. 5-HT biosynthesis and metabolism were explored in isolated eyestalks. The monoamine oxidase blocker pargyline, at concentrations between 0.8 and 10 mmol l-1, elicited a dose-dependent increase in 5-HT content. The biosynthesis of 5-HT in the crayfish eyestalk is suggested by the presence of its immediate precursor (5-OH-TRP) and by the suppression of 5-HT synthesis induced by m-hydroxybenzyl-hydrazine (m-HBH), a blocker of 5-OH-TRP decarboxylase. The presence of immunopositive cell bodies and axons was demonstrated using an anti-5-HT antiserum. 5-HT-like immunopositivity was detected in various regions of the eyestalk. Efferent immunopositive axons were also identified in the optic nerve, and these may have originated in the protocerebral lobe of the supraoesophageal ganglion. The branchings of these axons were profusely distributed in the neuropile of the medulla terminalis. A basal level release of 5-HT was detected in isolated eyestalks. The amount recovered was increased two-to threefold after blocking 5-HT uptake with fluoxetine (1 micromol l-1). Incubation of eyestalks in solutions containing a high K+ concentration (80 mmol l-1) released 5-HT. Electrical stimulation of the optic nerve released 5-HT as a function of the intensity of stimulation. Both the basal and evoked release were suppressed by lowering the Ca2+ concentration in the medium. These observations support a role for 5-HT as a neurotransmitter or neuromodulator in the crayfish eyestalk.

Journal Article↗

Inhibition of human chorionic gonadotrophin-induced hypertrophy of the ovaries and uterus in immature mice by some pineal indoles, 6-hydroxymelatonin and arginine vasotocin.

Administration of the indoles 5-methoxytryptophol, 6-hydroxymelatonin, melatonin, N-acetylserotonin or 5-methoxyindole-3-acetic acid (six 100-mug injections given at intervals of 12 h) inhibited both the absolute and relative ovarian and uterine weights of immature female mice pre-treated with HCG. Administration of 5-hydroxytryptophol or 5-methoxytryptamine at the same dosage inhibited ovarian but not uterine hypertrophy in mice pre-treated with HCG.

Animals↗

Effect of pineal indoles on testicular histology of mice.

The effect of late afternoon injections of melatonin, 5-methoxytryptamine, 5-methoxytryptophol, and 5-methoxyindole-3-acetic acid on testicular histology in mice were examined. Melatonin and 5-methoxytryptophol injections caused a reduction in the diameters of seminiferous tubules. The tests of melatonin-treated animals underwent some detectable regressive changes in the seminiferous tubules, whereas administration of 5-methoxytryptamine or 5-methoxytryptophol appeared to cause atrophy in some tubules. The percentage of aspermic tubules in melatonin-treated and methoxytryptamine-treated mice was significantly higher than that of the control. In involuted testes, some seminiferous tubules contained only Sertoli cells together with spermatogonia and spermatocytes, but no discernible spermatids and spermatozoa. Regressing spermatids and cell debris were frequently observed in the tubules. The tested of mice that received daily injections of 5-hydroxytryptophol and 5-methoxyindole-3-acetic acid were indistinguishable from those of the controls.

Animals↗

A study of indoles which inhibit pineal antigonadotrophic activity in male hamsters.

Blinding adult male golden hamsters led to involution of the testes and accessory sex organs (seminal vesicles and coagulating glands) and to a regression in pituitary prolactin levels within 8 weeks. The subcutaneous implantation of either melatonin or 6-hydroxymelatonin (1 mg/wk in beeswax) prevented the atrophy of the reproductive organs and the decrease in the stores of pituitary prolactin. Two other indoles, N-acetylserotonin and 5-hydroxytryptophol, failed to counteract the reproductive effects of blinding. Both melatonin and 6-hydroxymelatonin significantly elevated plasma LH titers.

Animals↗

Hereditary thrombocytopathies with abnormal release reaction (author's transl).

The authors have examined 8 cases with Hermansky-Pudlak syndrome in whom besides the usual abnormalities of abnormal aggregation with collagen, absence of second wave of aggregation, reduction of the 5-HT uptake, presence of 5-HIAA in the platelets, two new abnormalities are described: the presence of a large amount of an unidentified metabolite after 5-HT incorporation which differs from 5-HT and 5 hydroxytryptophol and an abnormal incorporation of labelled glycerol in the triglycerides. Correlation between abnormal lipid metabolism and defective 5-HT incorporation is discussed.

Adenosine Diphosphate↗

Time course and reproducibility of urinary excretion profiles of ethanol, methanol, and the ratio of serotonin metabolites after intravenous infusion of ethanol.

BACKGROUND: The aim of this study was to determine the time course and reproducibility of urinary excretion profiles of ethanol, methanol, and ratio of serotonin metabolites, 5-hydroxytryptophol (5HTOL) to 5-hydroxyindoleacetic acid (5HIAA), under strictly controlled conditions. METHODS: Nine healthy volunteers (6 women and 3 men) received 0.40 g of ethanol/kg of body weight on two occasions by constant rate intravenous infusion 30 min. Urine was voided before administration of ethanol and thereafter every 60 min for a total of 8 hr. Concentrations of ethanol and methanol in urine were determined by headspace gas chromatography, and the serotonin metabolites 5HTOL and 5HIAA were measured by gas chromatography-mass spectrometry and high-performance liquid chromatography, respectively. RESULTS: The peak concentration of ethanol in urine occurred at 30 or 60 min after the infusion ended. The peak concentration of methanol and the 5HTOL/5HIAA ratio developed more gradually, reaching a plateau at 213 to 220 min and 200 to 220 min postinfusion, respectively. The concentration-time profiles of ethanol and the 5HTOL/5HIAA ratio varied more between subjects than within subjects (p < 0.001), whereas the inter- and intraindividual variation in the pharmacokinetics of methanol were not significantly different (p > 0.05). The concentration of methanol and the 5HTOL/5HIAA ratio remained above the endogenous levels at 8-hr postinfusion despite the fact that urinary ethanol was no longer measurable (<0.01 g/liter) after 5 to 6 hr. CONCLUSIONS: Measuring the concentrations of methanol and the ratio of serotonin metabolites (5HTOL/5HIAA) in urine is a more sensitive way to monitor recent drinking, compared with analysis of ethanol in body fluids. Moreover, the concentration-time profiles of methanol and the 5HTOL/5HIAA ratio showed a good test-retest stability. The present intravenous infusion experiment confirms previous work on these markers when ethanol was given perorally. The urinary methanol and the 5HTOL/5HIAA ratio have applications in forensic and clinical medicine as indicators of acute alcohol consumption.

Adult↗

[Diagnostic tests of alcohol consumption].

Alcoholism is one of the most frequent dependences. In the cases of excessive alcohol consumption laboratory tests become abnormal and, therefore, may have ability to detect alcohol-dependent subjects. We present the biological markers for recent alcohol intake such as ethanol, methanol and 5-hydroxytryptophol and the most obvious and specific tests for chronic alcohol consumption including gamma-glutamyltransferase, aspartate and alanine aminotransferase, carbohydrate-deficient transferrin, macrocytosis, beta-hexosaminidase and erythrocytic aldehyde dehydrogenase.

Alanine Transaminase↗

Calcium dependent regulation of catecholamine and serotonin metabolism in human neuroblastoma cells.

Three human neuroblastoma cell lines were shown to have markedly different contents of catecholamines and serotonin. Two of the cell lines (CHP-134 and IMR-5) have higher levels of dopamine and its metabolites, while CHP-404 cells have higher levels of serotonin and its metabolites. Each cell line responded to the addition of D,L-2-amino-5-phosphonovalerate, an agent which increases plasma membrane permeability to Ca2+ (Pastuszko and Wilson, 1988; with striking changes in the metabolism of the neurotransmitters. These changes were dependent on the extracellular calcium concentration and include activation of dopamine synthesis (tyrosine hydroxylase), increased levels of dihydroxyphenylacetic acid and increased formation of N-methylated dopamine derivatives. Catabolism of serotonin to 5-hydroxyindole acetic acid was inhibited while that to 5-hydroxytryptophol was stimulated. These data clearly identify several important sites for regulation of neurotransmitter metabolism by calcium. The mechanisms, direct or indirect, by which the enzyme activities are modulated by calcium remain to be established.

2-Amino-5-phosphonovalerate↗

Radioimmunoassay of serotonin (5-hydroxytryptamine) in cerebrospinal fluid, plasma, and serum.

We describe a direct radioimmunoassay for serotonin (5-hydroxytryptamine) in cerebrospinal fluid, platelet-poor plasma, and serum. We raised antisera in rabbits against serotonin diazotized to a conjugate of bovine albumin and D,L-p-aminophenylalanine. Polyethylene glycol, alone or in combination with anti-rabbit immunoglobulins, is used to separate bound and unbound tritiated serotonin. The minimum concentration of serotonin detectable is 2 nmol/L in a 200-microL sample. Within-day precision (CV) is 4.3%, between-day precision 7.7%. Analytical recoveries of serotonin are 109% and 101% for cerebrospinal fluid and plasma, respectively. Tryptophan, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol do not interfere with the assay. However, 5-methoxytryptamine and tryptamine cross react. Of samples of cerebrospinal fluid from patients with disc herniations (n = 21) or low-pressure hydrocephalus (n = 10), one-third had concentrations of 2-4 nmol/L and two-thirds were below the minimum detectable concentration. The observed range for the concentration of serotonin in plasma of 14 normal subjects was 5-14 nmol/L (mean +/- SD, 9 +/- 3 nmol/L). The observed ranges for serotonin in serum were: for 10 women 520-900 (mean +/- SD: 695 +/- 110) nmol/L and for 10 men 380-680 (520 +/- 94) nmol/L.

Adult↗