Profiling of tryptophan-related plasma indoles in patients with carcinoid tumors by automated, on-line, solid-phase extraction and HPLC with fluorescence detection.
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BACKGROUND: Profiling of the plasma indoles tryptophan, 5-hydroxytryptophan (5-HTP), serotonin, and 5-hydroxyindoleacetic acid (5-HIAA) is useful in the diagnosis and follow-up of patients with carcinoid tumors. We describe an automated method for the profiling of these indoles in protein-containing matrices as well as the plasma indole concentrations in healthy controls and patients with carcinoid tumors. METHODS: Plasma, cerebrospinal fluid, and tissue homogenates were prepurified by automated on-line solid-phase extraction (SPE) in Hysphere Resin SH SPE cartridges containing strong hydrophobic polystyrene resin. Analytes were eluted from the SPE cartridge by column switching. Subsequent separation and detection were performed by reversed-phase HPLC combined with fluorometric detection in a total cycle time of 20 min. We obtained samples from 14 healthy controls and 17 patients with metastasized midgut carcinoid tumors for plasma indole analysis. In the patient group, urinary excretion of 5-HIAA and serotonin was compared with concentrations of plasma indoles. RESULTS: Within- and between-series CVs for indoles in platelet-rich plasma were 0.6-6.2% and 3.7-12%, respectively. Results for platelet-rich plasma serotonin compared favorably with those obtained by single-component analysis. Plasma 5-HIAA, but not 5-HTP was detectable in 8 of 17 patients with carcinoid tumors. In the patient group, platelet-rich plasma total tryptophan correlated negatively with platelet-rich plasma serotonin (P = 0.021; r = -0.56), urinary 5-HIAA (P = 0.003; r = -0.68), and urinary serotonin (P <0.0001; r = -0.80). CONCLUSIONS: The present chromatographic approach reduces analytical variation and time needed for analysis and gives more detailed information about metabolic deviations in indole metabolism than do manual, single-component analyses. (+info)
Biochemical studies on the mechanism of difference in the renal toxicity of 5-hydroxy-L-tryptophan between Sprague Dawley and Wistar rats.
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The biochemical mechanisms of the renal toxicity of 5-hydroxy-L-tryptophan to rats were studied using Wistar and Sprague Dawley rats, which had different LD50 values. When the amino acid was injected intraperitoneally, Wistar rats, which had a low LD50 value of 5-hydroxy-L-tryptophan, excreted larger amonts of serotonin and smaller amounts of 5-hydroxyindole acetic acid into the urine than Spraque Dawley rats, which had a high LD50 value. The activity of renal aromatic L-amino acid decarboxylase was higher in Wistar rats than in Sprague Dawley rats, while the activity of renal aromatic amino acid transaminase was in an opposite relationship. The activity of renal monoamine oxidase was almost the same in both strains and the activity of renal UDP glucuronyltransferase in Wistar rats was higher than in Sprague Dawley rats. Since the renal damage caused in rats by 5-hydroxy-L-tryptophan was very similar to that caused by serotonin, the amine formed from the administered amino acid was thought to be an important factor for the renal necroses, and difference in serotonin formation from the administered precursor amino acid may be one of the important factors leading to the difference in LD50 values in the two strains of rats. (+info)
Quantitative analysis of tropomyosin linear polymerization equilibrium as a function of ionic strength.
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Tropomyosin is a coiled-coil protein that polymerizes by head-to-tail interactions in an ionic strength-dependent manner. We produced a recombinant full-length chicken alpha-tropomyosin containing a 5-hydroxytryptophan residue at position 269 (formerly an alanine), 15 residues from the C terminus, and show that its fluorescence intensity specifically reports tropomyosin head-to-tail interactions. We used this property to quantitatively study the monomer-polymer equilibrium in tropomyosin and to calculate the equilibrium constant of the head-to-tail interaction as a function of ionic strength. Our results show that the affinity constant changes by almost 2 orders of magnitude over an ionic strength range of 50 mm (between I = 0.045 and 0.095). We were also able to calculate the average polymer length as a function of concentration and ionic strength, which is an important parameter in the interpretation of binding isotherms of tropomyosin with other thin filament proteins such as actin and troponin. (+info)
The pharmacological profile of (R)-3,4-dihydro-N-isopropyl-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carbo xamide, a selective 5-hydroxytryptamine(1A) receptor agonist.
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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-carbo xamide (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. (+info)
Depletion of 5-HT disrupts prepulse inhibition in rats: dependence on the magnitude of depletion, and reversal by a 5-HT precursor.
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The 5-HT(1A) agonist 8-OH-DPAT has been reported to disrupt prepulse inhibition (PPI) of the acoustic startle reflex after local administration into the raphe nuclei. Because it is likely that 8-OH-DPAT disrupted PPI by activation of somatodendritic inhibitory receptors, and thereby, via a decrease in 5-HT neurotransmission, we tested whether chronic, drug-induced, depletions of 5-HT have similar effects. Rats were drug-treated for three consecutive days and tested in a short PPI paradigm on day 4, and retested 2 h later, after acute saline or drug administration. Repeated treatment with the 5-HT synthesis inhibitor p-chlorophenylalanine methyl ester (PCPA; 160 mg/kg) produced a small, but significant, attenuation of PPI, and a large decrease in extracellular 5-HT levels in the hippocampus, as measured in independent microdialysis experiments. An even larger depletion of 5-HT was obtained by substituting the 3(rd) PCPA administration with the 5-HT releaser d-fenfluramine (10 mg/kg); this combined treatment nearly abolished PPI in the majority of animals. The involvement of 5-HT in the latter effects was confirmed by the finding that low doses of the 5-HT precursor 5-hydroxy-L-tryptophan reinstated PPI during retest. These data, together with recently published studies, provide strong evidence that pharmacologically-induced depletion of 5-HT disrupts PPI. (+info)
Modification of 5-hydroxytryptophan-evoked 5-hydroxytryptamine formation of guinea pig colonic mucosa by reactive oxygen species.
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We studied whether reactive oxygen species (ROS) generated by normal colonic mucosa affect 5-hydroxytryptophan (5-HTP)-evoked 5-HT formation (measured as the sum of 5-HT plus 5-hydroxyindole acetic acid (5-HIAA) accumulation) of guinea pig's isolated colonic mucosa. Catalase (3000-6000 U/ml), a hydrogen peroxide (H2O2) scavenger or diphenylene iodonium (DPI, 10-100 microM), an NADPH oxidase inhibitor, concentration-dependently caused an increase of the sum of 5-HT plus 5-HIAA accumulation in the presence of 5-HTP (10 microM), but these drugs did not significantly affect the 5-HT-metabolite in the colonic mucosa measured as the ratio of 5-HIAA/5-HT. Exogenously applied H2O2 (10-100 microM) concentration-dependently inhibited the sum of 5-HT plus 5-HIAA accumulation. In contrast, neither superoxide dismutase (SOD, 100-300 U/ml), superoxide anion scavenger, nor dimetyl sulfoxide (1-5%, DMSO), a hydroxyl radical scavenger affected the sum of 5-HT plus 5-HIAA accumulation. Moreover, mucosa ROS generation was estimated using the chemiluminescence technique. SOD (100-300 U/ml), catalase (3000-6000 U/ml) or DPI (10-100 microM), concentration-dependently reduced luminol-enhanced chemiluminescence signal from the colonic mucosa, while allopurinol (10-100 microM), a xanthine oxidase inhibitor, did not affect the chemiluminescence signal. These results suggest that ROS is formed through an NADPH oxidase system in the guinea pig colonic mucosa, where it exerts a modulatory effect on mucosal 5-HT formation upon addition of 5-HTP. Thus, ROS formation from normal colonic mucosa could be considered to contribute to the control of 5-HT production in mucosa enterochromaffin cells. (+info)
DARPP-32 mediates serotonergic neurotransmission in the forebrain.
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Serotonin is implicated in the regulation of complex sensory, motor, affective, and cognitive functions. However, the biochemical mechanisms whereby this neurotransmitter exerts its actions remain largely unknown. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of molecular weight 32,000) is a phosphoprotein that has primarily been characterized in relation to dopaminergic neurotransmission. Here we report that serotonin regulates DARPP-32 phosphorylation both in vitro and in vivo. Stimulation of 5-hydroxy-tryptamine (5-HT4 and 5-HT6 receptors causes an increased phosphorylation state at Thr34-DARPP-32, the protein kinase A site, and a decreased phosphorylation state at Thr75-DARPP-32, the cyclin-dependent kinase 5 site. Furthermore, stimulation of 5-HT2 receptors increases the phosphorylation state of Ser137-DARPP-32, the casein kinase-1 site. Behavioral and gene transcriptional effects induced by compounds that selectively release serotonin were greatly reduced in DARPP-32 knockout mice. Our data indicate that DARPP-32 is essential not only for dopaminergic but also for serotonergic neurotransmission. (+info)
Kinetic compartment modeling of [11C]-5-hydroxy-L-tryptophan for positron emission tomography assessment of serotonin synthesis in human brain.
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The substrate for the second enzymatic step in serotonin synthesis, 5-hydroxy-L-tryptophan, labeled in the beta-position ([11C]-HTP), was used for positron emission tomography (PET) measurements in six healthy human participants, examined on two occasions. One- and two-tissue kinetic compartment modeling of time-radioactivity curves was performed, using arterial, metabolite-corrected [11C]-HTP values as input function. The availability of unchanged tracer in arterial blood plasma was > or = 80% up to 60 minutes after injection, while [11C]-hydroxyindole acetic acid and [11C]-serotonin accounted for the remaining radioactivity, amounting to < or = 16% and < or = 4%, respectively. Compartment modeling was performed for brain stem, putamen, caudate nucleus, anterior cingulate, white matter, and superior occipital, occipitotemporal, and temporal cortices. The average biologic half-life for plasma-to-tissue equilibrium was 7 to 12 minutes, and the volume of distribution was 0.2 to 0.5 microL.mL(-1). In all regions except white matter, the kinetic compartment model that included irreversible [11C]-HTP trapping showed significantly improved model fits with respect to a one-tissue compartment model. The [11C]-HTP trapping rate constant depended on the estimated tissue availability of the serotonin precursor tryptophan, known to reflect serotonin synthesis in healthy individuals, and correlated with serotonin tissue concentration and synthesis rates reported previously in literature. These findings suggest the use of [11C]-HTP PET measurements to investigate serotonin synthesis. (+info)