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A facile and efficient one-pot three-component synthesis of spiroazepinoindolones has been developed. Using DMF as solvent without any additional acid or metal catalysts, the three-component reaction of readily available isatins, indolamines, and Meldrum's acid affords a new class of spiroheterocycles in moderate yield.

Capillary electrophoresis (CE) has been investigated for the analysis of some neurotransmitters, dopamine (DA), 3-methoxytyramine (3-MT) and serotonin (5-hydroxytryptamine, 5-HT) at nanomolar concentrations in urine. Field-amplified sample injection (FASI) has been used to improve the sensitivity through the online pre-concentration samples. The cationic analytes were stacked at the capillary inlet between a zone of low conductivity - sample and pre-injection plug - and a zone of high conductivity - running buffer. Several FASI parameters have been optimized (ionic strength of the running buffer, concentration of the sample protonation agent, composition of the sample solvent and nature of the pre-injection plug). Best results were obtained using H(3)PO(4)-LiOH (pH 4, ionic strength of 80 mmol L(-1)) as running buffer, 100 μmol L(-1) of H(3)PO(4) in methanol-water 90/10 (v/v) as sample solvent and 100 μmol L(-1) of H(3)PO(4) in water for the pre-injection plug. In these conditions, the linearity was verified in the 50-300 nmol L(-1) concentration range for DA, 3-MT and 5-HT with a determination coefficient (r(2)) higher than 0.99. The limits of quantification (10 nmol L(-1) for DA and 3-MT, 5.9 nmol L(-1) for 5-HT) were 500 times lower than those obtained with hydrodynamic injection. However, if this method is applied to the analysis of neurotransmitters in urine, the presence of salts in the matrix greatly reduces the sensitivity of the FASI/CE-UV method.Therefore, a solid phase extraction (SPE) on a dedicated imprinted polymer (MIP) was developed to extract specific neurotransmitters, catecholamines, metanephrines and indolamines, from urine. Matrix salts were thus discarded after sample extraction on AFFINIMIP™ Catecholamine & Metanephrine (100mg) cartridge. Therefore, lower limits of quantification were determined in artificial urine (46 nmol L(-1) for DA, 11 nmol L(-1) for 3-MT and 6 nmol L(-1) for 5-HT).The application of this protocol MIP-SPE/FASI-CE-UV analysis of neurotransmitters in human urine gave rise to electropherograms with a very good base line and signal to noise ratios above 15.

Brain glycogen could be considered as an energy store for neuronal activity, with high relevance in epilepsies. We selected two lines of mice based upon their latency to methionine sulfoximine (MSO) dependent-seizures: MSO-Fast and MSO-Slow, and their neurochemical characterization was attempted in order to look for the mechanisms of epileptogeny. We determined the MSO effect on brain glycogen in the two selected lines and their eight parental strains, and on indolamines and catecholamines. The increase in brain glycogen content induced by MSO is significantly lower in MSO-Fast than in MSO-Slow. At the onset of seizures the degradation of accumulated glycogen was higher in MSO-Slow mice than in MSO-Fast ones. Moreover, a positive correlation was observed between the magnitude of latency toward MSO-induced seizures and brain glycogen content in the eight parental strains used for selection. A striking proportionality between the content of glycogen and 5-hydroxytryptamine (5-HT) was observed in cerebral cortices of both selected lines. However, the cortical 5-HT level is higher in MSO-Fast than in MSO-Slow, and it is significantly decreased at the onset of seizures in both lines. Brain glycogen content is implicated in the developed model of mice with different latency to MSO-dependent seizures: The higher the brain glycogen content, the longer the latency; and 5-HT is involved in the control of latency to seizures-induced by MSO in these two lines. Our model of MSO "sensitive" (MSO-Fast) and "resistant" (MSO-Slow) mice could lead to a better understanding of MSO mechanisms of epileptogenesis, and the relationship between epileptogenic and glycogenic MSO effects.

Hyperglycemia and ketoacidosis are the two most serious factors in acute metabolic complications of both type 1 and type 2 diabetes. Dysfunction of the central nervous system is a well-documented complication of diabetes. We and others have previously reported that acute or chronic diabetes in animal's results in altered brain neurotransmitter levels. In this study, we investigated the effects of acute (7 days) glucose-induced hyperglycemia and sodium acetoacetate (NaAcAc) or ammonium chloride (NH4Cl) induced acidosis on the level of indolamines (5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA)) as well as PKC-alpha expression/activity in discrete areas of rat brain. Glucose-induced (500 mg/kg, bw) hyperglycemic ( approximately 249 mg%) rats showed significant (p

The purpose of the present study was to evaluate the effect of 4-pregnen-17-hydroxy-3-one (A) and two steroids homologues: 3beta-acetoxy-5,16-pregnadien-20-one (B) and 3beta-acetoxy-16alpha-17alpha-epoxy-4-pregnen-20-one (C). Male Wistar rats were treated with o-cresol combined (A, B or C) steroids. Lipid peroxidation status as result of measurement reactive substances to thiobarbituric acid (TBARS) as well as serotonin (5-HT) and its precursor 5-hydroxytryptophan (5-HTP) were measured. The prostate glands were weighed, the 5alpha-reductase activity was determined. The animals treated with A, B, and C steroids showed a slight increase in both 5alpha-reductase activity and prostate size. 5-HT and 5-HTP levels did not change significantly, and TBARS showed an increase in the group treated with B steroid and a decrease in the A steroid group with significant differences in both groups (p0.05) versus control group. Results suggest that A steroid reduces TBARS in rat brain, perhaps as a result of the interaction between the testosterone unsaturated carbons and OH(-) groups with free radicals.

Arylalkylamine N-acetyltransferase (NAT) from the female colleterial glands of Periplaneta americana showed activity peaks at pH 6.0 and 9.5 and the pH profile changed during oogenesis. The left gland contained higher activity than the right gland but the right gland also contained recognizable activity. The patterns in activity change depended on the substrate used, tryptamine (TN) or serotonin (5-HT). When TN was used as the substrate, the alkaline peak was higher than the acidic peak. In contrast, when 5-HT was used, the acidic peak was much higher than the alkaline peak. This suggests that at least two NATs are present in this species that are specific to pH and substrate species. Of the four combinations of the two pH ranges and two substrate indolamines, the enzyme activity that showed a similar change to the oocyte maturation was obtained in the combination of pH 6.0 and TN. TN was actually detected in the colleterial glands by fluorescent measurements according to Hess and Uderfriend [J. Pharmacol. Exp., 127 (1959) 175-177]. It peaked on the 6th day of emergence, which corresponded to the first rise of oocyte length and yolk accumulation, whereas a small peak appeared in the phase of the second rise. TN, or more likely N-acetyl TN, may therefore be involved in the regulation of oocyte maturation which could be a novel mechanism in oocyte maturation.

The serotonin system has been implicated in the modulation of endocrine and behavioral components of reproduction. In this study, we examined endogenous hypothalamic indolamines during sexual differentiation and long-term effects of exogenous steroids during this time. In Experiment 1, Japanese quail were studied during the last half of embryonic development and early post-hatch. Samples were taken at embryonic day 10 (E10), E12, E14, E16, hatch (day 0), and days 3 and 5, post-hatch. Hypothalamic indolamines, including serotonin (5-HT) and its metabolite, 5-hydroxy indole acetic acid (5-HIAA) were measured by HPLC-EC detection. Females had relatively higher hypothalamic 5-HT at E14 than males, with both sexes showing increasing levels thereafter. By day 5, post-hatch, hypothalamic 5-HT content was higher in males than in females. When turnover was estimated by comparing relative concentrations of 5-HT to 5-HIAA, males were significantly higher at E12 and E14 than females. These data suggest that there are stage specific changes in the serotonin system, as well as sexually dimorphic patterns in the ontogeny and activity of this system. In Experiment 2, we investigated the effects of embryonic steroid hormone treatment on the serotonin system and on male sexual behavior. Birds were treated with either estradiol benzoate (EB), testosterone propionate (TP) or sesame oil (vehicle control) at selected embryonic days (E10, E12, E14, E16, 0, D3, and D5). At 4 weeks post-hatch, birds were transferred to short photoperiod (16D:8L) for 3 weeks to prevent photostimulated reproductive development. At 7 weeks of age, males were implanted with a 20 mm silastic capsule filled with testosterone and sexual behavior was tested 1 week later. Brains were collected from both males and females, and preoptic area (POA) indolamines were measured. Steroid treatment at E10 or E12 resulted in the loss of male sexual behavior. Moreover, males treated with EB or TP on E12 also had increased POA 5-HT content as adults, compared to control males. Females treated with EB on either E10 or E 12 also had higher POA 5-HT content than control or TP treated females. These data provide evidence for sexual dimorphism in the hypothalamic 5-HT system at specific stages during embryonic development. Moreover, males were sensitive to exogenous EB and TP on E12, whereas females appeared to be affected by EB only and appeared to be sensitive to steroid effects over a longer period of time in development. Moreover, exogenous steroids at E12 in males also correlated with impaired sexual behavioral. These data suggest that long-term effects of embryonic steroid exposure may be mediated in part through effects on the serotonin neurotransmitter system.

A serotonin N -acetyl transferase (NAT) activity was detected in the brain of Pieris brassicae . The maximal head NAT activity appeared to occur during the wandering-pharate phase. The haemolymph level of serotonin, the selected NAT substrate, presented a maximum on the 3rd day of the 5th instar for both short- and long-photoperiod animals with a longer increase in short-photoperiod animals. The circadian rhythm of head NAT, determined on the same day, appeared bimodal and was affected by pteridines only when they were injected at “specific” times. At the last larval stage, the effect of either serotonin, N -acetylserotonin or melatonin was characterized by inactivity immediately after the injection, leading to death by starvation. In “underchilled” chrysalids, serotonin induced a diapause breakdown while melatonin led to mortality. These data suggest, besides the role of juvenile hormone, ecdysone, and polypeptidic hormones, a role for indolamines during the photoperiodic-dependent development of Pieris .

The application of a tissue-based biosensor for the measurement of the concentration of catecholamines and indolamines is described. The bioelement of the biosensor is a rat liver section and the reaction of the deamination of biogenic amines (monoamineoxidase) was used. The concentration of NH + 4 was measured by using ah ammonium-selective electrode. The biosensor was used for measuring tyramine, dopamine, tryptamine and serotonin in the range ca. 10 −3 –10 −4 M; the lifetime of the biosensor is about 20–25 days. Optimization of the parameters of the biosensor is discussed.

1. Apart from octopamine, the same indolamines and catecholamines were detected in the whole nervous system of the cockroach Blaberus craniifer Burm., at the same time. 2. However, levels were found to depend on sex, age, and the anatomic region within the nervous system. 3. Although not established, it is thought that these substances act synchronously from the ganglia. 4. The differences in levels between males and females and between anatomical regions during imaginal life suggest, in this species of cockroach, the physiological importance of the metameric organization in metabolic pathways or functional aspects of biogenic amines.

1. Indolamine levels were determined in the cerebral ganglion, the thoraco-abdominal nerve cord (except the last ganglion), and the 6th abdominal ganglion of females of Blaberus craniifer . 2. Measurements were made at the imaginal molt and on fed and crowded imagos at 10, 20 and 30 post-imaginal days. 3. Indolamines were found in the nervous system of young females, but 5-hydroxytryptophan was only detected in the thoraco-abdominal nerve cord. 4. Amine levels were related to the age of the cockroach, particularly during this period, to post-ecdysi events and ootheca formation.

The effects of different doses of chronically administered carbaryl on rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during darkness (2300 h and 0100 h) when pineal melatonin synthesis is high were studied. Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Carbaryl was administered at total doses (over 6 days) of either 50, 125 or 250 mg/kg by gastric gavage. Control rats received vehicle (corn oil) only. During the study, the rats were exposed to light/dark cycles of 14:10 with lights off at 2100 h. Pineal NAT and HIOMT activities and pineal melatonin were increased at 0100 h following carbaryl administration at all three doses. Conversely, serum melatonin was increased at 2300 h after the 250 mg/kg dose of carbaryl while all three doses of the pesticide reduced serum melatonin levels at 0100 h. Pineal 5-HTP, 5-HT and 5-HIAA levels were usually increased at 2300 h but unaffected at 0100 h. The results indicate that carbaryl has significant effects on pineal melatonin synthesis and secretion.

Tetrahydrobiopterin (BI-L) is the cofactor for the pteridine-de~ndent monoxygenases (phenylalanine tyrosine and tryptophan hydroxylases) (Kaufmann and Fisher 1974), which play important roles in the biosynthesis of biogenic amines such as catecholamines and indolamines. The biosynthesis of these biogenic amines is regulated by the hydroxylat~on reaction of amino acids such as tyrosine and tryptophan (Nagatsu et al. 1964; Lovenberg et al. 1967), and this hydroxylation reaction is regulated by the concentration of BH4 (Kettler et ~J. !974). These biogenic amines, especially noradrenaliE.e and serotonin, have been thought to be related to affective disorders (Van Praag 1982). Therefore, the relationship between BH4 and depression has been of particular interest, and some controversial results about this relationship have been reported: lowering of the BI-L~ synthesi,~ in the postmortem brains of patients with depression (Blair et al. 1984); no difference between the CSF biopterin levels in patients with depression and in normal controls (Kellner et al. 1980; Levine and Lovenberg 1984); an in

Low MAO activity has been reported in a number of studies to be lower in patients with chronic schizophrenia (see recent review by WYATT and MURPHY,~ cotwins of schizophrenic@ as well as in patients with bipolar (manic depressive) disorders3 .Since MAO is an important enzyme in the degradation of both catecholamines and indolamines, low MAO in schizophrenics would be consistent with two of the leading conceptualizations of biochemical abnormalities in the major psychoses-the transmethylation and dopamine hypotheses. Further, platelet MAO activity is under genetic control as shown by studies of twins.4,5

The hypothalamus exerts an inhibitory influence on prolactin release from the anterior pituitary. Whether or not peptide hypothalamic prolactin-regulating factors, analogous to those for other pituitary hormones, exist, remains to be confirmed. There is evidence that catecholamines and indolamines directly or indirectly affect prolactin release, This concept may explain the pathogenesis of galactorrhœa-amenorrhœa and other endocrine diseases produced by hypothalamic-pituitary disorders.

p-Chloropherrylalaninc, a drug that selectively inhibits the synthesis of serotooin, has been used in order to invcstigate the localization of this amine in nerves of the pineal gland of rats. Glands of normal and treated rats were studied under the electron microscope, after fixing with osmium tetroxide and glutaraldehyde, and applying specific histochemistry for catechol and indolamines. Fluorometric determinations of scrotonin, noradrenalinc and dopaminc were also made in normal and treated glands. In normal glands about 35 % of granulated vesicles were observed in nerve endings fixed with osmium tetroxide or glutaraldehyde. After treatment with p-chlorophenylalanine the dense core of the granulated vesicles disappeared in glands fixed with glutaraldehyde but remained unchanged or increased in number in those fixed in osmium tetroxide. p-Chlorophcnylalanine produced an 87% decrease in serotonin content of the pineal gland while noradrenalinc and dopamine content did not diminish, The correlation between morphological and biochemical data suggests that both catechol and indolamines are stored in granulated vesicles in the pineal nerves of the rat. This assumption was confirmed with the specific histochemical technique of wood to differentiate catechol and indolamines.

Summary Drugs that may change the metabolism of catecholamines and indolamines have been used in order to investigate the nature of the granulated vesicles found in adrenergic nerve endings min the pineal gland of the rat. In normal endings, 30–40 per cent of the vesicles are granulated and the rest are homogeneous. The proportion of granulated vesicles diminish considerably between 2–48 hr after a single injection of reserpine and this is slowly restored to normal levels in a period of 8 days. A single injection or chronic treatment with iproniazid (a MAO inhibitor) increases the proportion of granulated vesicles, and also the size of the vesicles and granules. Iproniazid prior to reserpine protects the nerve endings from the depletion of granulated vesicles. Pyrogallol (a COMT inhibitor) does not change the concentration of granulated vesicles and does not protect the nerve ending from the action of reserpine. These results are interpreted as supporting the concept that the granulated vesicles contain the adrenergic transmitter. The possible mechanism of action of these drugs on the submicroscopic structure of adrenergic nerve endings is discussed.