Radioenzymatic microassay for picogram quantities of serotonin or acetylserotonin in biological fluids and tissues

This paper describes several modifications of the original radioenzymatic assay for serotonin which increase the sensitivity of the assay 20-fold as well as enhance its reliability. Using this method serotonin concentrations can be directly measured in biological examples without precleaning the sample. When compared to currently available methods this assay is specific and sensitive to approximately 1 pg of serotonin and can be used to measure serotonin levels in individual brain nuclei or microliter quantities of biological fluids. This assay can be easily adapted for the direct measurement of N-acetylserotonin. A large number of samples can be assayed in a single working day.     

An automated assay for brain serotonin

This communication describes an automated assay for brain serotonin. The assay is an adaptation of a commonly used manual assay which utilizes the reaction of o-phthalaldehyde with serotonin to develop fluorescence (2). In the automated assay the samples to be analyzed were mixed with 7.5 N HCl and o-phthalaldehyde in the presence of reduced glutathione, heated at 80°C, cooled and their fluorescence recorded. Linearity with respect to serotonin was demonstrated from 0 to at least 5 μM, and the lower limit of sensitivity was 0.7 ng serotonin (0.4 ml of 0.01 μM). The % error (standard deviation times 100 divided by the mean) was always 2% or less. The specificity was studied, and the assay was applied to the measurement of rat brain serotonin levels by demonstrating an increase in brain serotonin levels after pargyline treatment and a decrease after reserpine treatment.     

A nonradioactive high-throughput/high-content assay for measurement of the human serotonin reuptake transporter function in vitro

Both the tricyclic and specific serotonin reuptake inhibitor classes of antidepressants act primarily by inhibiting the reuptake of released serotonin by the human serotonin reuptake transporter (hSERT). In this article, the authors describe the use of a fluorescent substrate of the transporter (4-(4-(dimethylamino)-styrl)-N-methylpyridinium, ASP) to develop a microplate-based high-throughput screen for hSERT function. The assay is sensitive to known inhibitors of serotonin uptake, including fluoxetine (Prozac), with the correct rank order of potency and IC(50) values close to those reported in the literature for tritiated serotonin uptake. The authors also describe the validation of the assay for natural product screening using a test set of 2400 pure phyto-chemicals and 80 plant extracts. The mean Z of the screened plates was 0.53. Hit rates, confirmation rates, and validation of the hits in a "classical" assay for serotonin uptake are all reported. The assay can also be read in "high-content" mode using a subcellular imaging device, which allows direct detection of possible assay interference by acutely cytotoxic compounds. Among the compounds identified were several previously reported inhibitors of the hSERT, as well as compounds having structural similarity to the tricyclic antidepressant drugs.     

Determination of 5-hydroxytryptophan, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid in 20 rat brain nuclei using liquid chromatography with electrochemical detection

High-performance liquid chromatography with electrochemical detection is utilized for the simultaneous determination of serotonin, its precursor 5-hydroxytryptophan, and its major metabolite 5-hydroxyindoleacetic acid in nervous tissue samples. Tissue preparation required only homogenization in acidic solution and centrifugation prior to application to the chromatograph. Detection limits in the low picogram range were obtained for those indoles separated. This assay was used in combination with a micropunch dissection technique of 20 discrete rat brain nuclei to measure serotonin, its precursor, and major metabolite. The specificity of the assay was checked with pharmacological experiments aimed to increase or decrease serotonin levels. Pargyline, a monoamine oxidase inhibitor, led to a marked increase in serotonin and a decrease of 5-hydroxyindoleacetic acid while p-chlorophenylalanine, by blocking the conversion of tryptophan to 5-hydroxytryptophan, selectively depleted 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid.     

A rapid non-destructive fluorometric assay for gangliosides

An assay for gangliosides has been developed which is both rapid and non-destructive. The procedure is based on specific ganglioside serotonin binding and utilizes the inverse relationship between serotonin dialysis rates and ganglioside concentrations. The amount of serotonin in the diffusate after 30 minutes of dialysis is measured fluorometrically and converted, via a standard curve, to ganglioside content. Samples containing as little as 10 nanomoles of ganglioside have been assayed. A single assay is complete in 30 minutes and triplicate assays require less than an hour. Since no hydrolysis or other chemical reaction is involved, samples can be recovered intact by further dialysis and lyophilization.     

Microbore liquid chromatography with dual electrochemical detection for the determination of serotonin and 5-hydroxyindoleacetic acid in rat brain dialysates

A microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin and its metabolite 5-hydroxyindoleacetic acid in rat brain dialysates. The concentration of serotonin in these samples is usually in the low nanomolar range (fmol per 20 μl range). To optimize separation and detection, several adaptations were made to the system with respect to the injection valve, flow-rate of the pump, connections between injector, column and detector, and cell volume of the detector. These aspects are discussed, as well as the procedure developed for optimal peak identification of serotonin and correct estimation of 5-hydroxyindoleacetic acid. The assay allows the measurement of basal serotonin release without the use of a re-uptake inhibitor added to the perfusion fluid.     

Fractionation of simian virus 40 DNA fragments by RPC-5 column chromatography

This report describes several modifications of the original radioenzymatic assay for serotonin (4) which increase the sensitivity of the assay 10-fold as well as enhance its reliability. Serotonin is converted to [³H]melatonin, in two steps. First, serotonin is acetylated to N-acetylserotonin by acetic anhydride. The N-acetylserotonin is then incubated with hydroxyindole-O-methyltransferase and S-[methyl-³H]adenosyl methionine and is converted to [³H]melatonin. The radioactive melatonin is extracted with toluene-isoamyl alcohol (7:3), dried, reconstituted, isolated by one-dimensional, silica gel, thin-layer chromatography, and counted in a liquid scintillation counter. The assay is specific and sensitive to approximately 5 pg of serotonin and thus can be used to measure serotonin levels in single brain nuclei or microliter quantities of biological fluids. The assay can be easily adapted for the direct measurement of N-acetylserotonin. A large number of samples can be assayed in a single working day.     

Intrathecal serotonin in mice: analgesia and inhibition of a spinal action of substance P

Serotonin, administered intrathecally in mice, produced dose-related analgesia in the tail flick test and the subcutaneous hypertonic saline assay. Low doses (2.5-5 ng) of serotonin blocked the biting and scratching response elicited by intrathecal substance P. However, higher doses of serotonin itself elicited a behavioral syndrome characterized by scratching of the torso with the hindlimbs. Both the analgesic response and the scratching response due to serotonin were blocked by specific serotonin antagonists and the analgesia is likely mediated by a postsynaptic action on dorsal horn nociceptive neurons.     

Orcadian Rhythm of Serotonin Binding in Rat Brain—I. Effect of the Light-Dark Cycle

High affinity serotonin binding to rat brain membranes showed a circadian rhythm with minimal binding at 1000 and a maximal binding at 0000. Brain serotonin levels were almost inverse to the rhythm of serotonin binding. Under reverse light-dark conditions, lights on from 1900 to 0700, a significant phase shift in serotonin binding and concentration of about 8-10 hr was found. The adaptation of the rats to the inverse light-dark cycle was ascertained by plasma ACTH and corticosterone assay.     

Effects of starvation and neuroactive drugs on feeding in Caenorhabditis elegans

Caenorhabditis elegans concentrates its food, bacteria, by pharyngeal pumping. The rate of pumping is affected by the presence of bacteria. Using a new assay that allows measurement of pumping rate in a population of worms suspended in liquid by measuring their uptake of microscopic iron particles, we have confirmed and quantitated this effect. Furthermore, we demonstrated that starvation stimulates pumping. Worms that had been deprived of bacteria for more than 4 hours pumped in the absence of bacteria under conditions in which well-fed worms did not. Furthermore, starved worms responded to lower amounts of bacteria than did fed worms. The assay was also useful for measuring effects of drugs on pumping. Of about 30 chemicals screened, 5 had clear effects. The neurotransmitter serotonin and the serotonin uptake inhibitor imipramine stimulated pumping, while the serotonin antagonist gramine inhibits. Imipramine stimulation is greatly decreased in cat-1 and cat-4 mutants, which have low levels of serotonin. Muscimol, an agonist for the neurotransmitter GABA, and ivermectin, whose site of action may also be the GABA receptor, both inhibit pumping. Qualitative observations suggested a role for acetylcholine in the regulation of pumping.     

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

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

A modified enzymatic-isotopic microassay for serotonin (5HT) using 5HT-N-acetyltransferase partially purified from Drosophila

Serotonin-N-acetyltransferase (EC 2.3.1.5), partially purified from $\text{Drosophila}$$\text{melanogaster}$ (DNAT) was substituted for rat liver enzyme (RNAT) in a previously published radioenzymatic assay for serotonin (5HT). The purpose was to determine if this modification would increase the sensitivity and reliability of the original assay. Compared with RNAT, DNAT is 3–4 times more active; it lacks secondary 5HTP decarboxylase activity; and it is more stable. Under conditions of the modified assay, the only radioactive product formed from hypothalamic tissue extracts is ³H-melatonin; which can be measured from as little as 15 pg of serotonin substrate. Thus, substitution of DNAT for RNAT improves the original radioenzymatic assay allowing measurement of endogenous 5HT in hypothalamic nuclei from individual animals.     

Effects of 5,6-Dihydroxytryptamine on the Release, Synthesis, and Storage of Serotonin: Studies Using Rat Brain Synaptosomes

5,6-Dihydroxytryptamine is a neurotoxic analogue of serotonin which can have profound cardiovascular effects within minutes of administration in vivo (Korner and Head, 1981). These effects have been attributed to 5,6-dihydroxytryptamine-induced serotonin release, although there has been no biochemical assessment of the extent to which this occurs. The present study utilized an in vitro synaptosomal assay to determine the short-term effects of 5,6-dihydroxytryptamine on endogenous serotonin release, synthesis, storage, and metabolism. 5,6-Dihydroxytryptamine produced a rapid depletion of serotonin. At lower concentrations of 5,6-dihydroxytryptamine (0.1-1 microM), this depletion was associated primarily with an increase in the levels of 5-hydroxyindoleacetic acid, the deaminated metabolite of serotonin, with small increases in the amount of serotonin release. At higher concentrations (10-100 microM), a greater proportion of the depleted serotonin was released with less metabolism occurring. When metabolism was prevented by inhibiting monoamine oxidase, the amount of serotonin which was released equalled the amount of serotonin depletion. Thus monoamine oxidase activity was important in controlling the amount of serotonin which could be released by 5,6-dihydroxytryptamine. Further studies demonstrated that an impairment in serotonin synthesis and vesicular storage could account for the rapid depletion produced by 5,6-dihydroxytryptamine. Taken together, the results indicate that 5,6-dihydroxytryptamine acts to displace serotonin from vesicular stores into the cytoplasm where it can either be deaminated by monoamine oxidase or be released. Moreover, it is hypothesized that the intraneuronal concentration of 5,6-dihydroxytryptamine is important in determining the extent of serotonin release, because it can inhibit the deamination of serotonin by monoamine oxidase.     

Biochemical and ultrastructural study of the disruption of blood platelets by streptolysin O

The membrane-damaging protein toxin, streptolysin O, proved highly lytic on human, guinea-pig and rabbit platelets. About 15 molecules of toxin were sufficient to lyse one cell. Platelet disruption was assessed by electron microscopy, clearing of cell suspensions and assay of lactate dehydrogenase, serotonin, monoamine oxidase and glutathione peroxidase released in the extracellular fluid. This egress reflected the damage of both plasmic and organelle membranes. A quantitative study of lactate dehydrogenase and serotonin liberation taken as respective markers of the cytosol and dense bodies was undertaken as a function of toxin concentration. No platelet aggregation or shape change was elicited by streptolysin O. The ghosts resulting from platelet lysis retained properties of the native membrane such as aggregability and serotonin uptake. Dense bodies were easily separated after gentle disruption of the plasmic membrane by small amounts of toxin. Platelet lysis by streptolysin O proved a useful procedure for the determination of protein content, enzyme activities and serotonin assay on the same lysate in contrast to usual methods.     

A simplified microassay for serotonin: Modification of the enzymatic isotopic assay

A simplification of the enzymatic isotopic assay for serotonin is described, Serotonin is converted to [³H]melatonin by a two-step reaction: N-acetylation of serotonin using acetic anhydride, followed by O-methylation with the enzyme hydroxyindole O-methyltransferase (EC 2.1,1.4) and S-adenosyl- -[methyl-³H]methionine as methyl donor. The present assay avoids the use of unstable acetylating enzyme, rat liver N-acetyltransferase (EC2.3,1.5). Blank values are lowered considerably and the sensitivity is doubly increased. Two-tenths micromole of serotonin per 30 μl of sample in tissue homogenates can be measured.     

Diurnal rhythms of serotonin in monkey cerebrospinal fluid

Serotonin concentrations in lumbar and ventricular cerebrospinal fluid (CSF) from rhesus monkeys showed a diurnal variation as measured by a gas chromatographic-mass spectrometric assay. Temporally, CSF serotonin and melatonin diurnal patterns were similar, with concentrations of both rising at the beginning of the dark period and falling to baseline at the onset of lighting. The amplitude of the nighttime serotonin elevations showed marked variation across the seven monkeys studied. A single monkey studied for three consecutive days had temporally and quantitatively consistent melatonin and serotonin concentrations.     

Serotonin Control of Thermotaxis Memory Behavior in Nematode Caenorhabditis elegans

Caenorhabditis elegans is as an ideal model system for the study of mechanisms underlying learning and memory. In the present study, we employed C. elegans assay system of thermotaxis memory to investigate the possible role of serotonin neurotransmitter in memory control. Our data showed that both mutations of tph-1, bas-1, and cat-4 genes, required for serotonin synthesis, and mutations of mod-5 gene, encoding a serotonin reuptake transporter, resulted in deficits in thermotaxis memory behavior. Exogenous treatment with serotonin effectively recovered the deficits in thermotaxis memory of tph-1 and bas-1 mutants to the level of wild-type N2. Neuron-specific activity assay of TPH-1 suggests that serotonin might regulate the thermotaxis memory behavior by release from the ADF sensory neurons. Ablation of ADF sensory neurons by expressing a cell-death activator gene egl-1 decreased the thermotaxis memory, whereas activation of ADF neurons by expression of a constitutively active protein kinase C homologue (pkc-1(gf)) increased the thermotaxis memory and rescued the deficits in thermotaxis memory in tph-1 mutants. Moreover, serotonin released from the ADF sensory neurons might act through the G-protein-coupled serotonin receptors of SER-4 and SER-7 to regulate the thermotaxis memory behavior. Genetic analysis implies that serotonin might further target the insulin signaling pathway to regulate the thermotaxis memory behavior. Thus, our results suggest the possible crucial role of serotonin and ADF sensory neurons in thermotaxis memory control in C. elegans.     

Role of serotonin in the regulation of human natural killer cell cytotoxicity

Addition of serotonin to mixtures of target cells and natural killer (NK)-enriched human mononuclear cells (MNC) in a 4-hr 51Cr-release assay strongly augmented NK cell cytotoxicity (NKCC) vs K562, Chang, or Molt-4 target cells. The effect was dose dependent at serotonin concentrations of 10(-4) to 10(-7) M, expressed at several effector to target cell ratios, and required the presence of accessory monocytes. A 5-HT1-specific receptor agonist, 8-OH-DPAT, mimicked the enhancing properties of serotonin with similar potency. Equimolar concentrations of the mixed 5-HT1/5-HT2 receptor antagonist cyproheptadine, but not the 5-HT2-specific antagonist ketanserin, completely blocked the serotonin-induced NKCC enhancement. Monocyte/NK cell mixtures incubated with serotonin for 1 hr produced a soluble factor that could enhance the cytotoxicity of autologous, NK-enriched cells depleted of monocytes, which did not respond to serotonin alone. The factor displayed no IFN or IL 2 activity as judged by the lack of antiviral activity and inability to support the growth of an IL 2-dependent cell line. In the presence of monocytes, serotonin (10(-5) M) was considerably more effective than human IFN-alpha or IFN-gamma at optimal concentrations and was about equally effective as IL 2 at a final concentration of 50 U/ml in a short-term NK assay. The potency and efficacy for serotonin were similar to that earlier reported for histamine in monocyte-containing effector cells. The NKCC-enhancing effect of serotonin was additive to that induced by IFN-alpha, IFN-gamma, or IL 2, but not to histamine. The presented data suggest an earlier unrecognized, serotonin receptor-mediated regulation of human NK cells.     

Effects of novel aggregation inhibitors on serotonin uptake by human blood platelets

Novel aggregation inhibitors blocked serotonin uptake by human blood platelets in concentrations ranging from 0.7 +/- 0.1 microM to 237.5 +/- 35.7 microM; a modified procedure, validated by kinetic analysis, was employed in which pH drift was minimized to 0.03 during the active assay period. Structural features in carbamoylpiperidine and nipecotoylpiperazine derivatives which actually constitute molecular probes, and show remarkable specificity for aggregation-inhibitory target sites, disclosed striking differences between the latter and serotonin receptors or other loci affecting serotonin uptake.     

Serotonin inhibition of synaptic transmission: Galpha(0) decreases the abundance of UNC-13 at release sites

We show that serotonin inhibits synaptic transmission at C. elegans neuromuscular junctions, and we describe a signaling pathway that mediates this effect. Release of acetylcholine from motor neurons was assayed by measuring the sensitivity of intact animals to the acetylcholinesterase inhibitor aldicarb. By this assay, exogenous serotonin inhibited acetylcholine release, whereas serotonin antagonists stimulated release. The effects of serotonin on synaptic transmission were mediated by GOA-1 (a Galpha0 subunit) and DGK-1 (a diacylglycerol [DAG] kinase), both of which act in the ventral cord motor neurons. Mutants lacking goa-1 G(alpha)0 accumulated abnormally high levels of the DAG-binding protein UNC-13 at motor neuron nerve terminals, suggesting that serotonin inhibits synaptic transmission by decreasing the abundance of UNC-13 at release sites.