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Positron emission tomography (PET) supplies a range of labeled compounds to be used for the characterization of tumor biochemistry. Some of these have proved to be of value for clinical diagnosis, treatment follow up, and clinical research. The first routinely used PET tracer in oncology, (18)F-labeled deoxyglucose (FDG), was successfully used for diagnosis of cancer, reflecting increased expression of glucose transporter in cancerous tissue. This tracer, however, usually does not show sufficient uptake in well-differentiated tumors such as neuroendocrine tumors. We developed a tracer more specific to neuroendocrine tumors-the serotonin precursor 5-hydroxytryptophan (5-HTP) labeled with (11)C-and demonstrated increased uptake and irreversible trapping of this tracer in carcinoid tumors. The uptake was so selective and the resolution was so high that we could detect more liver and lymph node metastases with PET than with CT or octreotide scintigraphy. To further improve the method, especially to reduce the high renal excretion of the tracer producing streaky artifacts in the area of interest, we introduced premedication by the decarboxylase inhibitor carbidopa, leading to a six-fold decreased renal excretion while the tumor uptake increased three-fold, hence improving the visualization of the tumors. (11)C-labeled l-DOPA was evaluated as an alternative tracer, especially for endocrine pancreatic tumors, which usually do not demonstrate enhanced urinary serotonin metabolites. However, only half of the EPTs, mainly functioning tumors, could be detected with l-DOPA. Instead 5-HTP seems to be a universal tracer for EPT and foregut carcinoids. With new, more sensitive PET cameras, larger field of view and procedures for whole-body coverage, the PET examination with 5-HTP is now routinely performed as reduced whole-body PET examinations with coverage of the thorax and abdomen. With this method we have been able to visualize small neuroendocrine lesions in the pancreas and thorax (e.g., ACTH-producing bronchial carcinoids) not detectable by any other method, including octreotide scintigraphy, MRI, and CT. Another tracer, the 11beta-hydroxylase inhibitor, metomidate labeled with (11)C, was developed to simplify diagnosis and follow-up of patients with incidentalomas. A large series of patients with incidentally found adrenal masses have been investigated and so far all lesions of adrenocortical origin have been easily identified because of exceedingly high uptake of (11)C-metomidate, whereas noncortical lesions showed very low uptake. In addition, adrenocortical cancer shows high uptake, suggesting that this PET tracer can be used for staging purposes.

Dopa-responsive dystonia (DRD) is an extrapyramidal disorder caused by deficit of 5,6,7,8-tetrahydrobiopterin (BH4), cofactor for tyrosine hydroxylase (TH). In these patients the nigrostriatal dopaminergic neurons normally express TH and the cellular machinery for the dopamine uptake. LA-N-1 is a human neuroblastoma cell line expressing tyrosine hydroxylase. Here we show that LA-N-1 cells are able to take up exogenous dopamine (DA) by an high-affinity mechanism; significant amounts of serotonin and its metabolite 5HIAA, but neither DA nor its metabolites, DOPAC and HVA, could be measured in the cell culture homogenate. 5,6,7,8-Tetrahydrobiopterin, cofactor for both tyrosine and tryptophan hydroxylases, is able to activate dopamine synthesis and also decreases the content of 5HIAA by 50%, indicating that LA-N-1 might be a useful model for studying dopamine-serotonin interaction in cultured cells and the neuronal mechanism of DRD.

Objective: The effects of repeated-restraint stress on brain 5-hydroxytryptamine; serotonin (5-HT) metabolism and functional responses to a selective 5-HT-1A agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), are compared in water- and ethanol-treated rats., Method: Locally bred male water- or ethanol-treated rats restrained 2 hours per day for 6 days were killed, and whole brains were collected for the neurochemical analysis by high performance liquid chromatography with electrochemical detection (HPLC-EC). In a separate experiment 8-OH-DPAT was injected in water- and ethanol-treated rats to compare elicited hyperactivity syndrome (a postsynaptic response) and effectiveness of the drug in reducing brain 5-HT synthesis (a presynaptic response)., Results: A single episode of 2-hour restraint stress decreased 24-hour cumulative food intake in both water- and ethanol-treated rats. Following repeated restraint stress of 2 hours per day for 5 days, the decreases were present in ethanol- but not water-treated rats. The sixth episode of 2-hour restraint stress did not alter brain tryptophan 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in water-treated repeatedly restrained rats but decreased tryptophan and increased 5-HT concentration in ethanol-treated rats. Ethanol-treated unrestrained and ethanol-treated repeatedly restrained rats exhibited higher levels of tryptophan 5-HT and 5-HIAA than their respective water-treated controls. Injecting 8-OH-DPAT at a dose of 0.25 mg/kg elicited comparable hyperactivity syndrome in water- and ethanol-treated rats but decreased 5-HT synthesis more in ethanol-treated than in water-treated rats., Conclusions: The present study shows that ethanol administration for 2 to 3 weeks, although it increases brain 5-HT metabolism, impairs adaptation by increasing the effectiveness of negative feedback control over 5-HT synthesis.

This study reports the presence of noradrenergic (NA) neurons which are capable to take up 5-hydroxytryptophan (5-HTP) and decarboxylate it to 5-hydroxytryptamine (5-HT serotonin) in the kitten brain. After loading of 5-HTP and monoamine oxidase inhibitor (MAOI), we could demonstrate 5-HT-immunoreactivity (IR) not only in hypothalamic and midbrain dopaminergic (DA) cell bodies, but also in NA ones located in the pons and medulla oblongata of the new born kitten aged from 1 to 7 days. NA cell bodies could no longer show 5-HT-IR after this treatment in the kitten older than 1 month. On the other hand, 5-HT-IR in the ventrolateral posterior hypothalamic (VLPH) cells was very weak at birth and became more and more intense after 15 days of age. Finally, after loading of tryptophan (TP) and MAOI, 5-HTP uptake cells mentioned above did not express 5-HT-IR in the kitten brain.

(S)-UH-301 ((-)-(S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin hydrochloride) is a well known 5-HT(1A) receptor antagonist. The present study describes the pharmacokinetic properties of (S)-UH-301 after subcutaneous administration in rats, using a newly developed HPLC-UV bioanalytical method. The relationships between (S)-UH-301 concentrations and some pharmacodynamic effects were also studied. The AUC of (S)-UH-301 in brain, but not in plasma, increased in proportion to dose (1-100 mumol/kg). However, at doses above 32 mumol/kg, peak concentrations of the drug did not increase in proportion to dose, and there was a doubling of its apparent half-life. There was a good correspondence between the time courses for the antagonism of 8-OH-DPAT-induced motor behaviours and hypothermia and the tissue concentrations of (S)-UH-301. Doses of (S)-UH-301 above 10 mumol/kg decreased 5-HT and dopamine synthesis. Therefore, a selective 5-HT(1A) antagonistic dose range of (S)-UH-301 should be 0.1-10 mumol/kg s.c., corresponding to concentrations below approximately 10 nmol/g in brain and approximately 1 nmol/ml in plasma.

The mechanisms by which fenfluramine suppresses food intake and body weight have been linked to its ability to enhance transmission across serotonin synapses in brain. This drug initially lowers body weight and suppresses food intake, yet after repeated administration food intake soon returns to normal and body weight no longer decreases. Fenfluramine also causes rapid and prolonged reductions in brain serotonin concentrations, which might account for its loss of appetite suppression. This possibility has been evaluated in rats by assessing if intermittent, chronic fenfluramine administration could suppress food intake during each treatment period, and if so, whether such an effect occurs in the presence of reduced brain serotonin levels. Rats were injected once daily with 10 mg/kg D,L-fenfluramine for 5 days, and then received no injections for the next 5 days. Control rats received only vehicle injections. This 10-day sequence was repeated five more times. During each period of fenfluramine administration, daily food intake dropped markedly the first 1-2 days of treatment, but returned to pretreatment values by day 5. Daily food intake was normal or slightly above normal during non-injection periods. Body weight dropped modestly during each period of fenfluramine administration, and rose during each subsequent period when injections had ceased. Serotonin concentrations and synthesis rates in several brain regions were markedly reduced at early, middle, and late periods of the experiment. Despite the long-term reduction in brain serotonin pools produced by fenfluramine, the drug continues to reduce food intake and body weight. Several possible interpretations of these findings are considered, based on the multiple mechanisms through which this drug has been proposed to modify synaptic serotonin transmission.

The activation of rostrally projecting serotonergic (5-HT) neurons by acute sound stress is blocked by exogenous administration of the tridecapeptide neurotensin (NT). 5-HT neurons respond to acute sound stress within the median raphe nucleus (MRN), but not within the dorsal raphe nucleus or hindbrain regions. By use of the NT antagonist, SR 48692, the present study examines the involvement of endogenous NT in modulating the preferential activation of MRN 5-HT neurons by sound stress, and extends the findings with sound stress to two other stressors (swim and tail shock). Activation is determined from the enhanced accumulation of 5-hydroxytryptophan (5-HTP) from various brain regions over basal after inhibition of aromatic amino acid decarboxylase. The NT antagonist, SR 48692, enhances the stress activation of MRN 5-HT neurons and its projections without changing 5-HTP accumulation under basal conditions. Thus, the antagonist, SR 48692, unmasks the action of endogenous NT-containing neurons indicating that they become activated by stress and serve to attenuate the stress-induced response of MRN 5-HT neurons.

The pharmacological properties of the 5-hydroxytryptamine (HT)(1A) receptor agonist (R)-3,4-dihydro-N-isopropyl-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide (NAE-086) were examined with in vitro and in vivo techniques. Receptor binding studies demonstrated that NAE-086 was a high-affinity and selective 5-HT(1A) receptor ligand with a K(i) value of 4.5 nM in membranes from rat hippocampus. Of 32 other receptors examined NAE-086 had a modest affinity only for the 5-HT(7) receptor (K(i) = 240 nM). NAE-086 inhibited VIP-stimulated adenylyl cyclase activity in GH(4)ZD10 cells with 79% of the efficacy of 5-HT. This inhibition was blocked by the 5-HT(1A) receptor (and beta-adrenoceptor) antagonist (-)alprenolol. A minor metabolite of NAE-086 in rats, (R)-3,4-dihydro-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide had a similar receptor profile but had 17 times higher affinity for the 5-HT(1A) receptor (K(i) = 0.26 nM). In vivo, NAE-086 induced all the typical effects of a 5-HT(1A) receptor agonist in rats: it decreased 5-HT synthesis (5-HTP accumulation) and 5-HT turnover (measured as the ratio of 5-hydroxyindoleacetic acid/5-HT), increased corticosterone secretion, induced the 5-HT(1A) syndrome (flat body posture and forepaw treading), inhibited the cage-leaving response, and caused hypothermia. All the responses mediated by postsynaptic 5-HT(1A) receptors were attenuated after single or repeated treatment of the rats with NAE-086. Simultaneously with the development of the tolerance to 5-HT(1A) receptor-mediated responses, 5-HT(2A) receptor-mediated responses were enhanced, as judged from the increased number of spontaneous and/or agonist [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]-induced wet-dog shake responses. The significance of this behavioral effect in relation to clinical observations is discussed.

DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan, respectively. DDC has been implicated in a number of clinic disorders, including Parkinson's disease and hypertension. Peripheral inhibitors of DDC are currently used to treat these diseases. We present the crystal structures of ligand-free DDC and its complex with the anti-Parkinson drug carbiDOPA. The inhibitor is bound to the enzyme by forming a hydrazone linkage with the cofactor, and its catechol ring is deeply buried in the active site cleft. The structures provide the molecular basis for the development of new inhibitors of DDC with better pharmacological characteristics.

The capillary networks of solid tumors are more permeable and less well organized than those of normal tissue. This review explores the hypothesis that these differences might be exploited as a selective antitumor strategy. Both high and low molecular weight compounds have been found to induce cytokines such as tumor necrosis factor (TNF) and to inhibit tumor blood flow in experimental tumors, with consequent induction of necrosis. Flavone acetic acid (FAA) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) are of particular interest. Accumulating evidence implicates the enzyme IkappaB kinase, which leads to the activation of the transcription factor, NFkappaB, as a target for these drugs. The downstream effects of FAA and DMXAA on the tumor microcirculation are complex, involving both direct and indirect effects on vascular endothelial cells. Induced changes in the shape and organization of vascular endothelial cells may lead to the activation of blood platelets and the release of 5-HT. Cytokines, 5-HT and nitric oxide (NO) released in response to FAA and DMXAA may induce a sustained increase in the permeability of tumor vascular cells, leading to cessation of blood flow and induction of tumor necrosis. Exploitation of this principle to clinical anticancer therapy represents an important challenge for the future.

High concentrations of the neurotransmitter serotonin can be found in inflamed and ischemic peripheral tissues, but the role of serotonin in immunoregulation is largely unknown. Here we report that serotonin protected human natural-killer (NK) cells from oxidatively induced inhibition inflicted by autologous monocytes in vitro. Serotonin protected NK cells from monocyte-mediated apoptosis and suppression of cytotoxicity and maintained the activation of NK cells induced by interleukin-2 despite the presence of inhibitory monocytes. A detailed analysis of these protective effects revealed that serotonin scavenged reactive oxygen species (ROS) derived from the H(2)O(2)-myeloperoxidase (-MPO) system. Serotonin shared this scavenger activity with its precursor, 5-hydroxytryptophan (5-HTP); however, serotonin was >10-fold more potent than 5-HTP in protecting NK cells against functional inhibition and apoptosis. We propose that serotonin, by scavenging peroxidase-derived ROS, may serve to protect NK cells from oxidative damage at inflammatory sites.

Acute electroconvulsive shock (ECS) causes a significant increase of protein synthesis in depressive patients and such an increase raises the possibility that the regulation of specific proteins and enzymatic activities in the brain might be one of the mechanisms required for the induction of long-term adaptive neurochemical changes after electroconvulsive therapy. In current studies, we investigated and compared simultaneously the short- and long-term effects of an acute ECS on the expression and enzymatic activities of both tyrosine and tryptophan hydroxylases (TH and TpOH, respectively) in different rat brain areas. Our results demonstrated that an acute ECS produced: (1) a long-lasting decrease in TH and TpOH protein levels in locus ceruleus (LC), ventral tegmental area (VTA) and in TpOH protein level in the raphe centralis (RC), maximal at 72 h, with concomitant changes in mRNA levels and enzymatic activities in the LC only; (2) large increase of TpOH protein levels in the frontal cortex (Cxf) (+145%) and increase of TH protein levels in the hippocampus (Hip) (+207%), maximal at 72 h and 7 days which was not accompanied by corresponding increase of in vivo enzymatic activities. Furthermore, a second ECS increased in vivo TpOH activity in the Cxf (+19%) while decreasing K(m) value (-50%) for tetrahydrobiopterin cofactor. A stability of the observed findings on TpOH activity in the Cxf after repeated ECS might be one of the mechanisms for the antidepressant effects of electroconvulsive therapy.

The serotonin syndrome results when serotonergic activity increases to abnormally high levels. It occurs with selective serotonin reuptake inhibitors (SSRIs), opioids, and other serotonergic agents when the serotonin system has been modulated by another serotonergic agent or compromised by illness. Although the symptoms are quite variable, the syndrome is characterized by a triad of altered mental status, neuromuscular abnormalities, and autonomic dysfunction. The authors report the probable occurrence of the serotonin syndrome with serotonin receptor subtype 3 (5-HT(3)) antagonist therapy when used to control nausea associated with chemotherapy in two seriously ill children. The first case involves combined use with mirtazapine and the second with fentanyl. These agents may pose a potential risk when used in such combination in seriously ill patients.

Mechanisms governing the effect of polychlorinated biphenyl (PCB) toxicity on hypothalamic serotonergic function and the neuroendocrine system controlling LH secretion were investigated in Atlantic croaker (Micropogonias unulatus) exposed to the PCB mixture Aroclor 1254 (1 microg x g body weight(-1) x day(-1)) in the diet for 30 days. PCB treatment caused a decrease in hypothalamic 5-hydroxytryptamine (5-HT) concentrations and significant inhibition of hypothalamic tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis, but did not alter the activity of monoamine oxidase, the catabolic enzyme. Further, PCB treatment caused significant decreases in GnRH content in the preoptic-anterior hypothalamic area. Significant decreases in pituitary GnRH receptor concentrations and the LH response to the GnRH analogue (GnRHa) were also observed in PCB-exposed fish, possibly as a consequence of a decline in GnRH release. The possible association between impaired serotonergic and neuroendocrine functions after PCB treatment was explored using serotonergic drugs. Treatment of croaker with p-chlorophenylalanine, an irreversible TPH inhibitor, mimicked the effects of PCB on the GnRH system and the LH response to GnRHa. Bypassing the TPH-dependent hydroxylation step with the administration of 5-hydroxytryptophan restored 5-HT to control levels and prevented the deleterious effects of PCB on the neuroendocrine parameters. Moreover, slow-release GnRH implants prevented the PCB-induced decline in GnRH receptors and restored the LH response to GnRHa, suggesting that GnRH therapy can reverse PCB-induced disruption of LH secretion. These results demonstrate that TPH is one of the targets of PCB neurotoxicity and indicate that a decrease in 5-HT availability in PCB-exposed croaker results in disruption of the stimulatory 5-HT/GnRH pathway controlling LH secretion.

The selective serotonin re-uptake inhibitor (SSRI) citalopram decreases the synthesis of 5-hydroxytryptamine (5-HT) in the mouse brain in vivo. The underlying mechanism was studied by recording the accumulation of 5-hydroxytryptophan (5-HTP) in hypothalamus and hippocampus after inhibition of the aromatic amino acid decarboxylase activity with m-hydroxybenzylhydrazine (NSD 1015). Depletion of 5-HT with reserpine markedly reduced the citalopram-induced decrease of 5-HTP but not that evoked by the 5-HT1A receptor agonist 8-OH-DPAT, which indicates that the presence of endogenous 5-HT is necessary for full effect of citalopram. In contrast to the almost complete antagonism of the decrease in 5-HT synthesis induced by 8-OH-DPAT, the 5-HT1A receptor antagonist WAY-100,635 only slightly affected the citalopram-evoked decrease in 5-HT synthesis. Likewise, the 5-HT1B receptor antagonists NAS-181 and GR127935 only slightly antagonised the citalopram effect although they strongly inhibited the decrease in 5-HT synthesis induced by the 5-HT1B receptor agonist anpirtoline. Combined treatment with 5-HT1A and 5-HT1B receptor antagonists did not produce any additive antagonistic effect on the citalopram-induced decrease in 5-HT synthesis. The 5-HT2A/2C receptor antagonist ketanserin, the 5-HT3 receptor antagonist ondansetron and the 5-HT4 receptor antagonist RS-39604 had no effect on the citalopram-induced decrease in 5-HT synthesis. The same was found for several other non-selective 5-HT receptor antagonists, e.g. cyproheptadine, dihydroergotamine, methiothepin, methysergide, metergoline and mianserin. It is concluded that the citalopram-induced decrease in 5-HT synthesis differs in sensitivity from that mediated by 5-HT1A or 5-HT1B receptor agonists and citalopram also seems to require endogenous 5-HT for its full effect.

A human midgut carcinoid tumor was successfully transplanted into nude mice and propagated for five consecutive generations (30 months) with well-preserved phenotype. Tumor cells in nude mice expressed identical neuroendocrine markers as the original tumor, including somatostatin receptors (somatostatin receptors 1 to 5) and vesicular monoamine transporters (VMAT1 and VMAT2). Because of the expression of somatostatin receptors and VMAT1 and VMAT2 the grafted tumors could be visualized scintigraphically using the somatostatin analogue 111In-octreotide and the catecholamine analogue 123I-metaiodobenzylguanidine. The biokinetics of the somatostatin analogue 111In-octreotide in the tumors was studied and showed a high retention 7 days after administration. Cell cultures were re-established from transplanted tumors. Immunocytochemical and ultrastructural studies confirmed the neuroendocrine differentiation. The human origin of transplanted tumor cells was confirmed by cytogenetic and fluorescence it situ hybridization analyses. Spontaneous secretion of serotonin and its metabolite, 5-hydroxyindole acetic acid, from tumor cells was demonstrated. The tumor cells increased their [Ca2+]i in response to beta-adrenoceptor stimulation (isoproterenol) and K+-depolarization. All somatostatin receptor subtypes could be demonstrated in cultured cells. This human transplantable carcinoid tumor, designated GOT1, grafted to nude mice, will give unique possibilities for studies of somatostatin receptor- and VMAT-mediated radionuclide uptake as well as for studies of secretory mechanisms.

The present study attempted to characterize the effects of electrolytic lesions of the hypothalamic dorsomedial nucleus on the daily profile of pineal metabolism as well as on the inhibition of pineal melatonin synthesis induced by acute light exposure during the night. Adult male Wistar rats (n = 107, 12:12 h light-dark cycle) were left intact (n = 47) or lesioned (n = 60). Lesioned rats and their respective controls were killed at six time points distributed throughout the light-dark cycle. At ZT (zeitgeber time) 18 the animals were killed either in the dark or after 15 min of light stimulation. Pineal glands were assayed using high-performance liquid chromatography with electrochemical detection (HPLC-ED). There was no difference in the amounts of pineal indoles between lesioned and control rats under any of the experimental situations tested. These results suggest that in rats, the hypothalamic dorsomedial nucleus does not participate in either the neural control of daily pineal metabolism or the nocturnal light-induced inhibition of the pineal metabolism., (Copyright 2001 S. Karger AG, Basel.)

Analysis of the reaction of dopa decarboxylase (DDC) with L-dopa reveals that loss of decarboxylase activity with time is observed at enzyme concentrations approximately equal to the binding constant, K(d), of the enzyme for pyridoxal 5'-phosphate (PLP). Instead, at enzyme concentrations higher than K(d) the course of product formation proceeds linearly until complete consumption of the substrate. Evidence is provided that under both experimental conditions no pyridoxamine 5'-phosphate (PMP) is formed during the reaction and that dissociation of coenzyme occurs at low enzyme concentration, leading to the formation of a PLP-L-dopa Pictet-Spengler cyclic adduct. Taken together, these results indicate that decarboxylation-dependent transamination does not accompany the decarboxylation of L-dopa proposed previously [O'Leary and Baughn (1977) J. Biol. Chem. 252, 7168-7173]. Nevertheless, when the reaction of DDC with L-dopa is studied under anaerobic conditions at an enzyme concentration higher than K(d), we observe that (1) the enzyme is gradually inactivated and inactivation is associated with PMP formation and (2) the initial velocity of decarboxylation is approximately half of that in the presence of O(2). Similar behaviour is observed by comparing the reaction with L-5-hydroxytryptophan occurring in aerobiosis or in anaerobiosis. Therefore the reaction of DDC with L-aromatic amino acids seems to be under O(2) control. In contrast, the reactivity of the enzyme with L-aromatic amino acids does not change in the presence or absence of O(2). These and other results, together with previous results on the effect exerted by O(2) on reaction specificity of DDC towards aromatic amines [Bertoldi, Frigeri, Paci and Borri Voltattorni (1999) J. Biol. Chem. 274, 5514-5521], suggest a productive effect of O(2) on an intermediate complex of the reaction of the enzyme with L-aromatic amino acids or aromatic amines.

In order to characterize the effects of naltrexone, a mu-opioid receptor antagonist, on acute ethanol-induced functional modification of dopaminergic neurons in the nigrastriatal and mesolimbic dopamine systems, the accumulation of L-3, 4-dihydroxyphenylalanine (L-DOPA) in the cerebral cortex, dorsal striatum and nucleus accumbens and of 5-hydroxy-L-tryptophan (5-HTP) in the hippocampus was measured in normal rats using the mu-hydroxybenzylhydrazine dihydrochloride (NSD-1015) enzymatic inhibition method. In addition, the firing rates of dopaminergic neurons were recorded in the substantia nigra and ventral tegmental area. Naltrexone resulted in a decrease in the dopaminergic neuronal firing rates activated by ethanol and eventually in a reduction of the dopamine synthesis induced by ethanol in the dorsal striatum and nucleus accumbens, but not in the cerebral cortex. Mesolimbic dopamine neurons were slightly more sensitive to ethanol and naltrexone than were nigrostriatal dopamine neurons. The widespread inhibitory action of naltrexone also decreased the ethanol-induced stimulation of hippocampal serotonin synthesis.

The effect of an endogenous 5-hydroxytryptamine (5-HT) precursor, 5-hydroxytryptophan (5-HTP), on the luminal outflow of 5-HT was examined using the luminally perfused isolated colon of the guinea pig, a model that would facilitate the pharmacological analysis of luminal 5-HT release from enterochromaffin cells (EC cells). 5-HTP (1-10 microM) concentration-dependently caused an increase of the luminal outflow of 5-HT. Either tetrodotoxin (0.3 microM) or atropine (0.2 microM) did not affect the 5-HTP-evoked increase in luminal 5-HT outflow, while the L-type calcium channel blocker, nicardipine (1 microM) or diltiazem (1 microM) reduced the 5-HTP-evoked 5-HT outflow by 47% and 61%, respectively. SB203186 (1 microM), a 5-HT4-receptor antagonist, enhanced the 5-HTP-evoked 5-HT outflow, while ramosetron (1 microM), a 5-HT3-receptor antagonist reduced the stimulating effect of 5-HTP by 66%. Ketanserin (0.1 microM), a 5-HT2A-receptor antagonist did not modify the stimulatory effect of 5-HTP. It is concluded that in the guinea pig colon, 5-HTP facilitates the luminal 5-HT release from EC cells, with no involvement of neuronal mechanisms and a non-neuronal cholinergic system. Furthermore, non-neuronal 5-HT3 and 5-HT4 receptors appear to contribute to the regulation of the luminal 5-HT release evoked by 5-HTP. This new bioassay of the guinea pig colon allows the pharmacological characterization of uncomplicated luminal 5-HT release from EC cells.

The autosomal recessive mutation dystonia musculorum (dt(J)/dt(J)) causes degenerative alterations of peripheral and central sensory pathways that lead to ataxia. To investigate possible changes in the central serotonin system of these mice, HPLC measurements of 5-hydroxytryptophan, 5-hydroxy-tryptamine (serotonin; 5-HT), and 5-HT metabolites were obtained from 22 brain regions and the spinal cord of wild type and dt(J)/dt(J) mutant mice. Also, 5-HT transporters were quantified by [(3)H]citalopram autoradiography in 72 brain regions, subregions, and nuclei, and the 5-HT innervation visualized by immunocytochemistry throughout the brain and spinal cord. In all brain regions measured for indoleamine content, there were no significant differences between the two genotypes. In the spinal cord, an increased tissue concentration of 5-HT (+34%), 5-hydroxyindole-3-acetic acid (+33%), 5-hydroxytryptophol (+21%), and 5-hydroxytryptophan (+45%) in dt(J)/dt(J) actually corresponded to the same total amount of each of these indoleamines in the entire spinal cord, when taking into account its reduced size in the mutants. Quantification of the binding to 5-HT transporters showed increases in the medial geniculate nucleus (+14%), medial (+24%) and lateral (+18%) hypothalamus, interpeduncular (+13%), vestibular (+22%), and deep cerebellar nuclei (+37%) of dt(J)/dt mice, and decreases in the ventral tegmental area (-13%), median and linear raphe nuclei (-20%), as well as in the solitary complex (-35%). There were no apparent differences in the distribution of 5-HT-immunostained fibers in these and other regions of brain and in the spinal cord of dt(J)/dt(J) compared to wild type mice. The bulk of these results indicates a relative sparing of the central 5-HT system in the dt(J)/dt(J) mice, even though alterations in 5-HT transporters could justify attempts at improving the sensorimotor dysfunction by administration of serotoninergic agents in these mice., (Copyright 2000 Wiley-Liss, Inc.)

The term for re-entrainment to a new light-dark cycle in streptozotocin (STZ)-induced diabetic rats was significantly longer than that in control rats. In STZ-induced diabetic rats, the same level of phase delay in the suprachiasmatic nucleus neuronal firing rhythm was observed in control rats after glutamate application. Therefore, 5-HT function in the hypothalamus is thought to be decreased in STZ-induced diabetic rats. These results suggest that postsynaptic neuronal function is still maintained in the suprachiasmatic nucleus of STZ-induced diabetic rats. Therefore, 5-HT mechanisms may play an important role in the maintenance of this function.

Indoleamine 2,3-dioxygenase, the first and rate-limiting enzyme in human tryptophan metabolism, has been implicated in the pathogenesis of many diseases. The human enzyme was expressed in Escherichia coli EC538 (pREP4) as a fusion protein to a hexahistidyl tag and purified to homogeneity in terms of electrophoretic and mass spectroscopic analysis, by a combination of phosphocellulose and nickel-agarose affinity chromatography. The yield of the fusion protein was 1.4 mg per liter of bacterial culture with an overall recovery of 56% from the crude extract. When the culture medium was supplemented with 7 microM hemin, the purified protein contained 0.8 mol of heme per mole of enzyme and exhibited an absorption spectrum consistent with the ferric form of hemoprotein. The pI value of the recombinant enzyme was 7.09 compared with 6.9 for the native enzyme. This was as expected from the addition of the hexahistidyl tag. Similar to the native enzyme, the recombinant enzyme required methylene blue and ascorbic acid for enzyme activity and oxidized not only l-tryptophan but also d-tryptophan and 5-hydroxy-l-tryptophan. The molecular activities for these substrates and their K(m) values were similar to those of the native enzyme, indicating that the addition of the hexahistidyl tag did not significantly affect catalytic activity. The recombinant protein can therefore be used to investigate properties of the native enzyme. This will aid the development of specific inhibitors of indoleamine 2,3-dioxygenase, which may be effective in halting disease progression., (Copyright 2000 Academic Press.)

We have examined using microdialysis the effect of intracranial self-stimulation (ICSS) on the in vivo hydroxylation rate of tyrosine and tryptophan in the medial prefrontal cortex (mPFC), nucleus accumbens (NAC) and striatum (STR). A decarboxylase inhibitor NSD-1015 was included in the perfusate, which enabled the simultaneous measurement of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) as an index of the in vivo hydroxylation level of tyrosine and tryptophan. When rats were exposed to 1 h of ICSS at the medial forebrain bundle (MFB), their extracellular levels of DOPA significantly increased in the mPFC, NAC and STR, but with a different magnitude and time course. The same stimulation produced a delayed increase in extracellular 5-HTP, compared to DOPA, in these brain regions. The profile of 5-HTP response demonstrated no apparent difference among the regions. These findings indicate that ICSS of the MFB can increase differentially the in vivo hydroxylation of tyrosine but similarly the in vivo hydroxylation of tryptophan in the mPFC, NAC and STR.

Our previous study demonstrated that intracranial self-stimulation of the medial forebrain bundle can increase the in vivo synthesis turnover rate of dopamine (DA) and serotonin (5-HT) in the nucleus accumbens of adrenal-intact rats. The present study examined using microdialysis whether such increases in DA and 5-HT syntheses are influenced by adrenal hormones, which are also activated following intracranial self-stimulation. A decarboxylase inhibitor, NSD-1015, was perfused through reversed microdialysis which enabled the simultaneous measurement of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) as an index of the in vivo turnover rate of DA and 5-HT syntheses. Adrenalectomy (ADX) attenuated significantly the self-stimulation-induced increase in dialysate levels of DOPA but not 5-HTP. Corticosterone (Cort) replacement reversed the attenuation in DOPA levels in adrenalectomized rats. The finding indicates that activation of DA synthesis in vivo in the nucleus accumbens during intracranial self-stimulation is dependent on, whereas that of 5-HT synthesis is independent of glucocorticoid modulation.

NAS-181 ((R)-(+)-2-(3-morpholinomethyl-2H-chromen-8-yl) oxymethyl-morpholine methanesulfonate) is a novel rat 5-hydroxytryptamine1B, (r5-HT1B) receptor antagonist with high selectivity. The in vivo effects of NAS-181 on 5-HT metabolism and synthesis in the rat brain were examined. 5-HT metabolism, measured as the ratio 5-hydroxyindoleacetic acid (5-HIAA)/5-HT, was dose-dependently increased in all four brain regions analysed (hypothalamus, hippocampus, frontal cortex and striatum) at doses 0.1 to 20 mg/kg s.c. NAS-181. The enhancement of 5-HT metabolism at the dose 20 mg/kg s.c. was maximal one hour after the injection and was still significant eight hours but not 24 hours after the injection. 5-HT synthesis rate measured as the accumulation of 5-hydroxytryptophan (5-HTP) after inhibition of the aromatic amino acid decarboxylase activity was also elevated by NAS-181 at doses 0.3 to 20 mg/kg s.c. NAS-181 competitively antagonised the decrease in 5-HT metabolism evoked by the r5-HT1B receptor agonist, anpirtoline, in hypothalamus, hippocampus and frontal cortex. Anpirtoline had no effect on 5-HT metabolism in striatum. However, anpirtoline antagonised the enhancement of 5-HT metabolism induced by NAS-181 in striatum. Combined treatment of rats with NAS-181 and the 5-HT1A receptor antagonist, WAY-100635, increased 5'-HT metabolism considerably more than when the compounds were given alone.

Levels of 5-hydroxytryptophan (5-HTP) in brain regions (hypopthalamus and medulla) of rainbow trout were analysed by HPLC-EC 0, 10, 30, and 40 min after intraperitoneal administration of different doses of L-tryptophan (Trp) (0, 12.5, and 25 mg. kg(-1) body weight) in fish treated with 3-hydroxybenzylhydrazine (NSD1015; 75 mg. kg(-1)). The results show that, in control fish, 5-HTP levels in hypothalamus (58.03 +/- 6.36 pg. mg(-1) brain tissue) were significantly higher than those observed in medulla (28.04 +/- 4.32 pg. mg(-1) brain tissue). Basal tryptophan hydroxylation rates (after 0 mg. kg(-1) Trp administration) were 0.42 +/- 0.07 pg 5-HTP. mg(-1). min(-1), and 0.63 +/- 0.24 pg 5HTP. mg(-1). min(-1), for hypothalamus and medulla respectively. On the other hand, the results demonstrate that L-tryptophan administration induced significant increases in the rate of tryptophan hydroxylation, both in hypothalamus and medulla. These findings indicate that, in a way similar to that observed in mammals, brain tryptophan hydroxylase is unsaturated by its substrate (tryptophan) under normal physiological conditions. J. Exp. Zool. 286:131-135, 2000., (Copyright 2000 Wiley-Liss, Inc.)

The present study examined the effect of systemic administration of lipopolysaccharide (LPS; 100 and 250 microg/kg, i.p.) on tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities in frontal cortex, striatum and midbrain of the rat. Enzyme activities were determined by measuring accumulation of the transient intermediates 5-hydroxytrptophan (5-HTP) and L-dihydroxyphenylalanine (L-DOPA) following in vivo administration of the decarboxylase inhibitor, NSD 1015. TPH activity was increased 2 hours after administration of LPS (100 and 250 microg/kg) in both frontal cortex and midbrain, and a secondary increase was seen in the midbrain 12 hours after challenge. LPS provoked an increase in TH activity in the midbrain only, and this was evident for up to 24 hours after LPS administration. Thus in addition to previous studies demonstrating that LPS increases in vivo NA, DA and 5-HT release, this study shows that LPS increases the activity of the rate-limiting enzymes responsible for their synthesis.

[1] We have previously shown that the beta-adrenergic/5-HT1 receptor partial agonist (-)-pindolol (2.0-32.0 micromol kg(-1)) enhances the increase in forward locomotion in rats produced by the 5-HT2 receptor agonist DOI (0.7 micromol kg(-1)) via net activation of post-synaptic 5-HT2 receptors. [2] It was found that neither the 5-HT1A receptor agonist and partial agonist, (+/-) 8-OH-DPAT (0.2-2.4 micromol kg(-1)) and (S)-(-)-UH-301, respectively, nor the 5-HT1A receptor antagonist WAY-100635 (0.09-1.5 micromol kg(-1)), substituted for (-)-pindolol in this in vivo behavioral model. [3] This also applies to the 5-HT1B receptor agonist and antagonist anpirtoline (0.3-4.0 micromol kg(-1)) and isamoltane (1.0-64.0 micromol kg(-1)), respectively. Neither of these compounds mimicked (-)-pindolol in its interactions with DOI. [4] The (-)-pindolol/DOI-induced increase in forward locomotion could be antagonized by the beta1 adrenoceptor antagonist betaxolol (24 micromol kg(-1)). [5] It is suggested that the intrinsic efficacy of (-)-pindolol at beta-adrenoceptors is an important aspect of its in vivo pharmacodynamic profile.

In three patients with carcinoid liver and/or lymph node metastases, we studied the process of tumor tracer uptake and decarboxylation by means of positron emission tomography (PET) using 5-hydroxy-L-tryptophan (5-HTP) 11C-labeled in the beta-position (HTP) and later the same day with 5-HTP 11C-labeled in the carboxyl group (HTC). With HTP, in which the 11C-label follows the molecule through decarboxylation to form 11C-serotonin, a high tumor accumulation of the tracer was found. With HTC, in which the label is rapidly eliminated from the tissues as 11CO2 if decarboxylation takes place, there was virtually no uptake by the tumors. By utilizing data from PET scanning with both tracers, we could quantify the decarboxylation rate and tissue accumulation of [11C]-serotonin and hence the enzymatic action of aromatic amino acid decarboxylase.

Objectives: We have previously reported that acute administration of the specific serotonin reuptake inhibitor (SSRI), fluoxetine, resulted in a decrease in mitogen-induced blood lymphocyte proliferation. The present studies further examine the specificity of this response to serotonin reuptake systems and the potential role of endogenous serotonin in mediating these effects., Methods: Male Sprague-Dawley rats were treated intraperitoneally with the SSRIs, fluoxetine (6-10 mg/kg) or sertraline (20 mg/kg), dopamine and norepinephrine reuptake inhibitors, GBR 12909 and desipramine, respectively (6 mg/kg) or the serotonin precursor, 5-hydoxytryptophan (5-HTP, 50 mg/kg) 2 h prior to sacrifice. The serotonin-depleting agents, p-chlorophenylalanine (PCPA, 2 x 200 mg/kg) or the serotonin-lesioning agent, p-chloroamphetamine (PCA, 2 x 10 mg/kg) were administered intraperitoneally 5-7 days prior to fluoxetine (10 mg/kg) administration., Results: Unlike the SSRIs, which significantly suppressed lymphocyte proliferation responses, selective norepinephrine or dopamine reuptake inhibition had no effect on lymphocyte proliferation. Elevation of extracellular serotonin levels with the serotonin precursor, 5-HTP, resulted in a similar decrease in lymphocyte proliferation as that seen with SSRI administration. Conversely, decreases in endogenous serotonin following PCA or PCPA treatment prevented the fluoxetine-induced decreases in lymphocyte proliferation., Conclusions: These results suggest that decreases in mitogen-induced lymphocyte proliferation following acute fluoxetine administration were due to elevations in extracellular serotonin following reuptake inhibition., (Copyright 2001 S. Karger AG, Basel)

The role of the serotoninergic system in acceleration of the sexual development of domesticated rats (Rattus norvegicus) was assessed. The onset of age-related changes in hypothalamic serotonin during prepubertal period occurred earlier in domesticated than in aggressive male rats. Blockade of the serotoninergic system after p-chlorophenylalanine (PCPA) administration on days 40 and 44 delayed the development of the reproductive system in both aggressive and domesticated males. In 60-day-old rats treated with PCPA, levels of testosterone in plasma and the number of mature spermatozoa in epididymis were decreased compared to controls. At the same time, the administration of PCPA on days 30 and 34 did not modify basal testosterone secretion and other parameters in 60-day-old aggressive rats and produced a decrease similar to PCPA injections on days 40 and 44, although less pronounced, in the weights of testes in domesticated animals. Administration of 5-hydroxytryptophan (5-HTP), a precursor of serotonin synthesis, on days 30, 32, 34, 36 and 38 increased plasma testosterone levels and weights of the sex organs in 60-day-old domesticated males, but did not significantly affect the development of reproductive system in aggressive animals. These data indicate that serotonin stimulates sexual development of males during prepubertal period and this activating effect of serotonin occurs earlier in domesticated than in aggressive males. They also suggest that the acceleration in sexual maturation of domesticated rats could result from changes in the ontogenetic dynamic of hypothalamic serotonin induced by a selection for low aggressiveness towards man.

We have introduced tryptophan codons at different positions of the chicken alpha-tropomyosin cDNA (Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466) and employed a trp auxotrophic Escherichia coli strain to express the proteins in media containing either normal tryptophan, 5-hydroxytrptophan, or 7-azatryptophan. The fluorescence of these latter two tryptophan analogues is excitable at 312-315 nm at which the natural fluorescence of other thin filament proteins (actin, troponin) is not excited. The recombinant tropomyosins have tryptophans or analogues located at amino acid positions 90, 101, 111, 122, or 185 of the protein, all on the external surface of the tropomyosin coiled-coil (positions "c" or "f" of the hydrophobic heptad repeat). The first four mutations are located within the third actin-binding zone of tropomyosin, a region not expected to interact directly with troponin or with neighboring tropomyosin molecules in muscle thin filaments, while position 185 is located in a region that has been implicated in interactions with the globular domain of troponin. The fluorescence intensity of the mutant containing 5-hydroxytryptophan at position 122 (5OH122W) is sensitive to actin binding and sensitive to Ca2+-binding to thin filaments reconstituted with troponin. Assuming that the globular domain of troponin binds to a site between residues 150 and 190 of tropomyosin, the distance between the troponin-binding site and the fluorescent probes at position 122 can be estimated to be 4.2-10.2 nm. While X-ray diffraction and electron micrograph reconstitution studies have provided evidence of Ca2+-induced changes in tropomyosin's interactions in the thin filament, their resolution was not sufficient to distinguish between changes involving the whole tropomyosin molecule or only that region directly interacting with troponin. Here we provide a clear demonstration that Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site which is probably associated with a changed interaction with actin.

The effects of antidepressant drugs on the synthesis of noradrenaline and serotonin (5-HT) were assessed using the accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition as a measure of the rate of tyrosine and tryptophan hydroxylation in the rat brain in vivo. Three inhibitory synthesis-modulating receptors were investigated simultaneously: the alpha2C-autoreceptor modulating dopa/noradrenaline synthesis, and the alpha2A-heteroreceptor and 5-HT1A-autoreceptor modulating 5-HTP/5-HT synthesis. Acute treatment (2 h, i.p.) with desipramine (1-10 mg/kg), protriptyline (0.3-10 mg/kg) and nisoxetine (3-10 mg/kg), selective NA reuptake blockers, dose-dependently decreased dopa synthesis in cortex (15%-40%) and hippocampus (20%-53%). Fluoxetine (1-10 mg/kg) and zimelidine (1-10 mg/kg), selective 5-HT reuptake blockers, did not alter dopa synthesis. Fluoxetine and zimelidine dose-dependently decreased 5-HTP synthesis in cortex (14%-43%) and hippocampus (27%-54%). Desipramine and protryptyline did not alter 5-HTP synthesis in cortex but in hippocampus it was decreased (36%). Repeated desipramine (10 mg/kg for 1-21 days) or fluoxetine (3 mg/kg for 3-21 days) treatment resulted in a time-dependent loss in their ability to decrease dopa or 5-HTP synthesis. Desipramine (1-21 days) did not alter 5-HTP synthesis in cortex, but in hippocampus it was decreased (21%-37%, days 1-14) followed by recovery to control values (day 21). Fluoxetine (3-21 days) did not alter brain dopa synthesis. To further assess the desensitization of alpha2C-autoreceptors, alpha2A-heteroreceptors and 5-HT1A autoreceptors regulating the synthesis of dopa/NA or 5-HTP/5-HT after chronic desipramine and fluoxetine, the effects of clonidine (agonist at alpha2-auto/heteroreceptors) and 8-OH-DPAT (agonist at 5-HT1A-autoreceptors) were tested. In saline-treated rats, clonidine (1 mg/kg, 1 h) decreased dopa and 5-HTP synthesis in cortex (58% and 54%) and hippocampus (54% and 42%). In desipramine-treated rats (10 mg/kg, 21 days), but not in fluoxetine-treated ones (3 mg/kg, 14 days), the effect of clonidine was attenuated in cortex (12% and 18%) and only for dopa synthesis in hippocampus (31%). In saline-treated rats, 8-OH-DPAT (1 mg/kg, 1 h) decreased 5-HTP synthesis in cortex (63%) and hippocampus (75%). In fluoxetine-treated rats, but not in desipramine-treated ones, this inhibitory effect was markedly attenuated in cortex (26%) and hippocampus (9%). These findings indicate that acute treatment with cyclic antidepressant drugs results in activation of inhibitory alpha2C-autoreceptors, alpha2A-heteroreceptors and/or 5-HT1A-autoreceptors regulating the synthesis of dopa/NA and/or 5-HTP/5-HT in brain, whereas chronic treatment with these drugs is followed by desensitization of these presynaptic receptors.

Carcinoid syndrome usually is associated with a classic presentation, the diagnosis of which is supported by elevations in neuroendocrine substances such as 5-hydroxyindoleacetic acid and by localization and pathologic identification of a neuroendocrine tumor. The authors report a patient for whom there was a 7-year delay in diagnosis and even then a primary tumor could not be localized.

We have used microdialysis to measure the in vivo hydroxylation level of tyrosine and tryptophan in the medial prefrontal cortex and nucleus accumbens of conscious rats that were subjected to immobilization. The brain was perfused with an inhibitor of aromatic L-amino acid decarboxylase, 3-hydroxybenzylhydrazine, and the amount of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) accumulating in the dialysate was measured as an index of the in vivo hydroxylation rate of tyrosine and tryptophan. One hour of immobilization caused a significant increase in extracellular DOPAin the medial prefrontal cortex but not nucleus accumbens. The same manipulation produced a significant and more prolonged elevation in extracellular 5-HTP in the nucleus accumbens as well as medial prefrontal cortex. The observed profile of stress-induced 5-HTP response was comparable in two brain regions. The results suggest that in vivo catecholamine synthesis is heterogenous, whereas in vivo serotonin synthesis is homogenous, with respect to responsiveness to stress in the medial prefrontal cortex and nucleus accumbens.

To study the mechanisms of antiepileptic action of zonisamide (ZNS), we determined the effects of ZNS on extracellular, total levels and re-uptake activity of serotonin (5-HT) in rat striatum and hippocampus. After acute administrations, plasma ZNS concentrations associated with anticonvulsive action (effective concentrations) increased the total levels of 5-HT, its metabolise (5-hydroxyindoleacetic acid: 5-HIAA) and precursor (5-hydroxytryptophan: 5-HTP). After chronic administration of ZNS, effective plasma concentrations also increased the extracellular and total levels of 5-HT, 5-HIAA, and 5-HTP. On the other hand, after both acute and chronic administrations of ZNS, a supra-effective ZNS concentration either decreased or did not affect the total levels of these substances. Therefore, the stimulatory effects of ZNS on the 5-HT system were reduced by an increase in ZNS concentration to supra-effective concentrations. ZNS concentrations of 30-1000 microM did not affect hippocampal 5HT re uptake activity in vitro. These results suggest that ZNS has biphasic effects on the 5-HT system, in that effective concentrations of ZNS enhance and supra-effective concentrations of ZNS reduce the function of the 5-HT system. These biphasic effects of ZNS on the 5-HT system may be involved in the mechanisms of action of the antiepileptic and psychotropic effects, and side effects of ZNS.

The objective was to examine effects of galaninrat on forebrain monoamine synthesis and on spontaneous locomotor activity in the rat. The rate of monoamine synthesis was estimated by measuring the accumulation of l-DOPA and 5-HTP, following inhibition of cerebral aromatic l-amino acid decarboxylase by means of NSD-1015 (100 mg kg-1 i.p.), after i.c.v. or intracerebral administration of galanin in adult male Wistar rats. Spontaneous locomotor activity was observed in an automated open-field arena ( approximately 0.5 m2). The i.c.v. administration of galanin (0.5-5.0 nmol bilaterally) produced a dose-dependent, statistically significant, increase in DOPA accumulation throughout the neostriatum, and in the olfactory bulb, indicating an increase in the rate of DA synthesis. No increase was observed in brain areas where noradrenaline is the predominant catecholamine, such as the neocortex or the ventral hippocampus. In addition, there was a tendency for an increase in 5-HTP accumulation in the dorso-lateral neostriatum and in the accumbens. The same i.c.v. administration of galanin produced a dose-dependent, and statistically significant, decrease in spontaneous locomotor activity. The effect on forebrain DA synthesis could also be produced by local bilateral application of galanin (2x1 nmol) into the ventral tegmental area, but not the nucleus accumbens (2x2 nmol). There were no effects on forebrain DOPA or 5-HTP accumulation by the local application of galanin into the locus coeruleus, or into the dorsal raphe nucleus. It is concluded that the neuropeptide galanin modulates forebrain dopaminergic neurotransmission. The effect appears to be mediated at the somato-dendritic level of the meso-neostriatal pathway, and could perhaps be utilized to normalize perturbations ascribed to dysfunction in this neuronal pathway, such as schizophrenia., (Copyright 1999 Elsevier Science B.V.)

1. The effects of treating groups of rats with four different anabolic androgenic steroids (AAS) (testosterone, nandrolone, methandrostenolone, and oxymetholone) on 5-hydroxytryptamine (5-HT) and dopamine (DA) neurones in different brain regions were examined. The AAS was injected six times with 1 week's interval and the rats were sacrificed 2 days after the final injection. 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured. The effect on DA and 5-HT synthesis rate was analysed as the accumulation of 3,4-dihydroxyphenyl-alanine (DOPA) and 5-hydroxytryptophan (5-HTP), respectively, after inhibition of the amino acid decarboxylase with NSD-1015 (3-hydroxy-benzylhydrazine dihydrochloride). Additionally, the monoamine oxidase (MAO) activity was analysed in the hypothalamus. 2. The DOPAC + HVA/DA ratio was increased in the striatum in all treatment groups. However, the synthesis rate of DA was significantly increased only in the methandrostenolone treated group. 3. The 5-HIAA/5-HT ratio was increased in all treatment groups in the hippocampus, in the frontal cortex in the methandrostenolone-treated animals and in the hypothalamus in the testosterone- and oxymetholone-treated rats, while the 5-HT synthesis rate was not affected by the AAS-treatments. 4. The MAO-A activity was increased in the oxymetholone-treated rats while the other treatment groups were unaffected. The MAO-B activity was not changed. 5. The results indicate that relatively high doses of AAS increase dopaminergic and 5-hydroxytryptaminergic metabolism in male rat brain, probably due to enhanced turnover in these monaminergic systems.

3,4-Dihydroxyphenylalanine (Dopa) decarboxylase is a stereospecific pyridoxal 5'-phosphate (PLP)-dependent alpha-decarboxylase that converts L-aromatic amino acids into their corresponding amines. We now report that reaction of the enzyme with D-5-hydroxytryptophan or D-Dopa results in a time-dependent inactivation and conversion of the PLP coenzyme to pyridoxamine 5'-phosphate and PLP-D-amino acid Pictet-Spengler adducts, which have been identified by high performance liquid chromatography. We also show that the reaction specificity of Dopa decarboxylase toward aromatic amines depends on the experimental conditions. Whereas oxidative deamination occurs under aerobic conditions (Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C. (1996) J. Biol. Chem. 271, 23954-23959; Bertoldi, M., Dominici, P., Moore, P. S., Maras, B., and Borri Voltattorni, C. (1998) Biochemistry 37, 6552-6561), half-transamination and Pictet-Spengler reactions take place under anaerobic conditions. Moreover, we examined the reaction specificity of nicked Dopa decarboxylase, obtained by selective tryptic cleavage of the native enzyme between Lys334 and His335. Although this enzymatic species does not exhibit either decarboxylase or oxidative deamination activities, it retains a large percentage of the native transaminase activity toward D-aromatic amino acids and displays a slow transaminase activity toward aromatic amines. These transamination reactions occur concomitantly with the formation of cyclic coenzyme-substrate adducts. Together with additional data, we thus suggest that native Dopa decarboxylase can exist as an equilibrium among "open," "half-open," and "closed" forms.

The Lurcher (Lc/+) mutant mouse is characterized by a considerable atrophy of the cerebellum due to a massive loss of cerebellar Purkinje and granule cells, as well as of neurons from the inferior olivary nucleus. In this study the effects of a therapeutic combination of amantadine, thiamine and L-tryptophan on the serotonin (5-HT) innervation was assessed in Lurcher mice by autoradiography, using [3H]citalopram to label 5-HT transporters. In wild type mice as well as in both saline-treated and drug-treated Lurcher mutants, [3H]citalopram binding remained unchanged in forebrain and brainstem regions. In the cerebellum, labelling of deep cerebellar nuclei (CBnuc) was about twofold higher than in the cortex (CBctx). In saline-treated Lurcher mutants compared to wild type mice, the densities of [3H]citalopram were 98% higher in CBctx, and 180% higher in CBnuc. In CBctx of drug-treated Lurcher mutants, transporter densities were 89% higher than in the wild type, but did not differ from the saline-treated Lurcher. In the CBnuc of the drug-treated Lurcher mutants, [3H]citalopram binding was 50% higher than in the saline-treated Lurcher group, and 320% higher than in wild type mice. The results show that 5-HT transporters, already upregulated in the CBnuc of Lurcher mutant mice, can be further increased by a pharmacological treatment, possibly altering the availability of 5-HT in some of its target areas.

Analysis of melatonin (MEL) in pineal cell cultures by enzyme linked immunosorbent assay showed its concentration was increased by fusaric acid (FA), a mycotoxin produced by Fusarium species and associated with toxic duck and ostrich feeds. Subsequent cell culture studies demonstrated the precursors of MEL, 5-hydroxytryptophan (5HTP) and serotonin (5HT), were also affected by FA as well as other Fusarium mycotoxins. Herein we describe a technique for the analysis of 5HTP and 5HT in pineal cell cultures using HPLC with electrochemical detection (EC), and report on the effects of FA alone and in combination with fumonisin B1 (FB1) and deoxynivalenol (DON) on the levels of these MEL precursors.

The receptor-mediated control of brain monoamine synthesis was used to examine the in vivo intrinsic efficacy of the 5-HT1A receptor antagonists NAD-299, S(-)-UH-301 and WAY-100,635. The rate of monoamine synthesis was estimated by measuring the accumulation of DOPA and 5-HTP in the ventral neostriatum and the ventral hippocampus in rats pretreated with an inhibitor of cerebral aromatic L-amino acid decarboxylase. S(-)-UH-301 (2.0-32.0 micromol kg(-1)), but not WAY-100,635 (0.08-1.2 micromol kg(-1)), produced a decreased 5-HTP accumulation in the neostriatum and in the hippocampus. The administration of NAD-299 (0.75-12.0 micromol kg(-1)) resulted in a slight increase in neostriatal, but not hippocampal, 5-HTP accumulation. Neostriatal DOPA accumulation was decreased by S(-)-UH-301, whereas treatment with WAY- 100,635 resulted in an increase. NAD-299 did not affect neostriatal DOPA levels. There were no effects by any of these agents on DOPA levels in the ventral hippocampus. It is concluded that S(-)-UH-301, but not WAY-100,635 or NAD-299, displays intrinsic efficacy at brain 5-HT1A and DA D2/3 receptors, whereas WAY-100,635 behaves as a DA D2/3 receptor antagonist. By this comparison, NAD-299 appears to be the most selective and specific 5-HT1A receptor antagonist.

To explore the usefulness of Caco-2 cells in the study of intestinal dopaminergic and 5-hydroxytryptaminergic physiology, we have undertaken the study of aromatic L-amino acid decarboxylase (AADC), catechol-O-methyltransferase (COMT) and type A and B monoamine oxidase (MAO-A and MAO-B) activities in these cells using specific substrates. The activity of these enzymes was also evaluated in isolated rat jejunal epithelial cells. The results showed that Vmax values (in nmol mg protein(-1) h(-1)) for AADC, using L-DOPA as the substrate, in rat jejunal epithelial cells (127.3+/-11.4) were found to be 6-fold higher than in Caco-2 cells (22.5+/-2.6). However, Km values in Caco-2 cells (1.24+/-0.37 mM) were similar to those observed in rat jejunal epithelial cells (1.30+/-0.29 mM). Similar results were obtained when AADC activity was evaluated using L-5HTP as substrate; in rat jejunal epithelial cells Vmax values (in nmol mg prot(-1) h(-1)) were found to be 5-fold that in Caco-2 cells (16.3+/-1.0 and 3.0+/-0.2, respectively), and Km values in Caco-2 cells (0.23+/-0.08 mM) were again similar to those observed in rat intestinal epithelial cells (0.09+/-0.03 mM). Caco-2 cells were not able to O-methylate dopamine, in contrast to rat jejunal epithelial cells (Vmax = 8.6+/-0.4 nmol mg protein(-1)(h-1); Km = 516+/-57 microM). Vmax values (in nmol mg protein(-1)(h-1)) for type A and B MAO in Caco-2 cells (19.0+/-0.6 and 5.4+/-0.6, respectively) were found to be significantly lower (P<0.05) than those in rat jejunal epithelial cells (46.9+/-3.1 and 9.6+/-1.2, respectively); however, no significant differences in the Km values were observed between Caco-2 and rat jejunal epithelial cells for both type A and B MAO. In conclusion, Caco-2 cells in culture are endowed with the synthetic and metabolic machinery needed to form and degrade DA and 5-HT, though, no COMT activity could be detected in these cells.

A continuous fluorometric assay for tryptophan hydroxylase activity based on the different spectral characteristics of tryptophan and 5-hydroxytryptophan is presented. Hydroxylation of tryptophan at the 5-position results in a large increase in the fluorescence of the molecule. The assay selectively monitors the fluorescence yield of 5-hydroxytryptophan by exciting the reaction mix at 300 nm. The rate of increase of the emission signal was found to be directly proportional to the enzyme concentration. Inner filter effects due to quinonoid dihydropterin accumulation were eliminated by the inclusion of a thiol reductant. Activity measured using this assay method was found to be the same as that determined by established discontinuous HPLC assay methods. The application of the assay to routine activity measurements and to steady-state determinations with the substrates tryptophan and tetrahydropterin is described., (Copyright 1999 Academic Press.)

Parkinson's disease (PD) is characterized by decreased striatal dopamine, but serotonin (5-HT) is also reduced. Because 5-HT decreases following a single levodopa injection, levodopa has been suggested to contribute to PD's serotonergic deficits. However, in a recent study, rat striatal serotonin levels were reported to increase following 15-day levodopa administration. To address this issue, we administered levodopa (50 mg/kg) to rabbits for 5 days, then measured serotonin, its precursors tryptophan and 5-hydroxytryptophan (5-HTP), and its major metabolite 5-hydroxyindole-acetic acid (5-HIAA) in striatum and CSF. Striatal serotonin and tryptophan were unchanged, while 5-HTP and 5-HIAA increased 4- and 7-fold, respectively. CSF 5-HTP and 5-HIAA were also significantly increased. In levodopa-treated animals, 5-HTP concentrations were moderately correlated (r = 0.679) between striatum and CSF, while weak correlations were present between striatal and CSF concentrations of both serotonin and 5-HIAA. These results suggest that repeated levodopa treatment increases striatal serotonin turnover without changing serotonin content. However, levodopa-induced alterations in striatal serotonin metabolism may not be accurately reflected by measurement of serotonin and 5-HIAA in CSF.

para-Chlorophenylalanine (PCPA, 100-200 mg/kg) was used as a pharmacological tool to characterize the 5-hydroxytryptamine (5-HT) involvement in the emesis occurring 24 hr after the administration of cisplatin (10 mg/kg) in the ferret. PCPA was effective to antagonize the initial 8 hr period of retching and vomiting, but potentiated the emesis that occurred during the remaining 8- to 24-hr observation period. Tissue samples removed from the brainstem at 24 hr post injection of cisplatin alone revealed an elevation of 5-HT, dopamine and homovanillic acid that was antagonized by the injection of PCPA. Cisplatin also induced increases in the urinary levels of 5-hydroxyindoleacetic acid that was similarly antagonized by PCPA. Results are discussed in terms of the relevance of 5-HT to the model of cisplatin (10 mg/kg)-induced emesis in the ferret compared to the problem of acute and delayed emesis in man. The residual or delayed phase of cisplatin-induced emesis may involve a 5-HT-independent mechanism.

Background: Withdrawal from long-term cocaine use is accompanied by symptoms resembling major depression. Because acute cocaine affects serotonin (5-HT) neurons, and 5-HT dysfunction is implicated in the pathophysiology of depression, we evaluated the effects to 5-HT agonists in rats withdrawn from repeated injections of cocaine (15 mg/kg i.p., b.i.d., 7 days) or saline., Methods: In the first study, prolactin (PRL) responses elicited by the 5-HT-releasing agent fenfluramine, the 5-HT1A agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), and the 5-HT2A/2C agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) were examined as indices of postsynaptic 5-HT receptor function. In a second study, specific responses induced by 8-OH-DPAT, namely inhibition of brain 5-HT synthesis and stimulation of feeding, were examined as correlates of 5-HT1A autoreceptor function., Results: Prior treatment with cocaine did not modify fenfluramine-evoked PRL release; however, the PRL secretory response to 8-OH-DPAT was blunted and the PRL response to DOI was potentiated after chronic cocaine treatment. Cocaine exposure did not alter the inhibitory effect of 8-OH-DPAT on 5-HT synthesis. 8-OH-DPAT-induced feeding was influenced by prior cocaine, but this effect was secondary to pronounced baseline hyperphagia in the cocaine-treated group., Conclusions: These data indicate that withdrawal from chronic cocaine renders specific subpopulations of postsynaptic 5-HT1A receptors subsensitive and 5-HT2A/2C receptors supersensitive. No evidence for cocaine-induced changes in 5-HT1A autoreceptor responsiveness was found. A survey of the literature reveals similarities in the profile of 5-HT dysfunction between rats withdrawn from cocaine and humans diagnosed with depression. We propose that withdrawal from chronic cocaine in rats may serve as a useful animal model of depressive disorders.