THE UPTAKE OF BIOGENIC MONOAMINES BY THE ISOLATED AURICLE OF THE SNAIL (HELIX POMATIA)

—The uptake of [³H]5HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the auricle of Helix pomatia was studied. When tissues were incubated at 25°C in media containing radioactive amines, tissue:medium ratios of about 49:1, 14:1 and 5:1 for 5-HT, dopamine, noradrenaline, and octopamine respectively were obtained after a 20–30 min incubation time. Tissues incubated at 25°C in media containing radioactive amines for 20–30 mins showed that almost all (96%) the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]octopamine or [³H]noradrenaline. The high tissue:medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, showed saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver–Burk plots, two uptake mechanisms for 5-HT at 25°C were resolved; the high affinity uptake process having a K_m1 value of 6.0 ± 10^?8m and a V_m1 value of 0.115 nmol/g/min while the lower affinity process had a K_m2 value of 1.04 ± 10^?6m and a V_m2 value of 0.66nmol/g/min. At 0°C a single uptake mechanism for 5-HT occurred which gave a K_m value of 5.02 ± 10^?8m and a V_m value of 0.0165 nmol/g/min. In the case of dopamine, the Lineweaver–Burk plot at 25°C showed a single uptake process with values for K_m and V_m of 1.55 ± 10^?7m and 0.086 nmol/g/min respectively. This process did not function at 0°C. The effect of various agents and ions upon the accumulation processes for all amines was also studied, and the data indicate that the same neurons probably accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the auricle is a mechanism for inactivating these substances at 25°C and that an uptake mechanism for 5-HT also functions at 0°C. The results are discussed from the point of view of 5-HT's being the cardioexcitatory substance in the snail heart.     

Transport of monoamine and amino acid neurotransmitters by primary astroglial cultures

The transport of [³H]l-glutamate, [³H]l-aspartate, [³H]-aminobutyric acid ([³H]GABA), [³H]dopamine, [³H]norepinephrine and [³H]5-hydroxytryptamine (³H-5-HT) was measured in primary astroglial cultures from newborn rat cerebral hemispheres. There was a high-affinity uptake with aK _m of 69.0 M for L-glutamate, 12.3 M forl-aspartate and 3.1 M for GABA. The uptake showed properties of high capacity with aV _max of 17.0 nmol·mg prot^–1·min^–1 forl-glutamate, 1.1 nmol·mg prot^–1·min^–1 forl-aspartate and 0.04 nmol·mg prot^–1·min^–1 for GABA. No high-affinity high capacity transport system was found for the monoamines studies. Autoradiographic examination demonstrated a heavy deposit of grains suggesting a prominent accumulation of [³H]l-glutamate and [³H]l-aspartate in the astroglial-like cells of the cultures, while the [³H]GABA accumulation was less intense. On the other hand, there was only a weak accumulation of grains after incubating the cultures with [³H]dopamine, [³H]norepinephrine or [³H]5-HT. Thus, astroglial cells in culture accumulate amino acid neurotransmitters and monoamines in different ways with a high-affinity high-capacity uptake of glutamate, aspartate and GABA and a diffusion-uptake of dopamine, norepinephrine and 5-HT.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

Safety and mechanism of appetite suppression by a novel hydroxycitric acid extract (HCA-SX)

A growing body of evidence demonstrates the efficacy of Garcinia cambogia-derived natural (–)-hydroxycitric acid (HCA) in weight management by curbing appetite and inhibiting body fat biosynthesis. However, the exact mechanism of action of this novel phytopharmaceutical has yet to be fully understood. In a previous study, we showed that in the rat brain cortex a novel HCA extract (HCA-SX, Super CitriMax) increases the release/availability of radiolabeled 5-hydroxytryptamine or serotonin ([³H]-5-HT), a neurotransmitter implicated in the regulation of eating behavior and appetite control. The aim of the present study was 2-fold: (a) to determine the effect of HCA-SX on 5-HT uptake in rat brain cortex in vitro; and (b) to evaluate the safety of HCA-SX in vivo. Isolated rat brain cortex slices were incubated in oxygenated Krebs solution for 20 min and transferred to buffer solutions containing [³H]-5-HT for different time intervals. In some experiments, tissues were exposed to HCA-SX (10 M – 1 mM) and the serotonin receptor reuptake inhibitors (SRRI) fluoxetine (100 M) plus clomipramine (10 M). Uptake of [³H]-5-HT was expressed as d.p.m./mg wet weight. A time-dependent uptake of [³H]-5-HT occurred in cortical slices reaching a maximum at 60 min. HCA-SX, and fluoxetine plus clomipramine inhibited the time-dependent uptake of [³H]-5-HT. At 90 min, HCA-SX (300 M) caused a 20% decrease, whereas fluoxetine plus clomipramine inhibited [³H]-5-HT uptake by 30%. In safety studies, acute oral toxicity, acute dermal toxicity, primary dermal irritation and primary eye irritation, were conducted in animals using various doses of HCA-SX. Results indicate that the LD₅₀ of HCA-SX is greater than 5000 mg/kg when administered once orally via gastric intubation to fasted male and female Albino rats. No gross toxicological findings were observed under the experimental conditions. Taken together, these in vivo toxicological studies demonstrate that HCA-SX is a safe, natural supplement under the conditions it was tested. Furthermore, HCA-SX can inhibit [³H]-5-HT uptake (and also increase 5-HT availability) in isolated rat brain cortical slices in a manner similar to that of SRRIs, and thus may prove beneficial in controlling appetite, as well as treatment of depression, insomnia, migraine headaches and other serotonin-deficient conditions.     

Characterization of Multiple [3H]5–Hydroxytryptamine Binding Sites in Rat Spinal Cord Tissue

Abstract: High-affinity [³H]5-hydroxytryptamine ([³H]5-HT) binding in the rat spinal cord is similar to that demonstrated in the frontal cortex. [³H]5-HT binds with nearly the same affinity to sites in both tissues. Furthermore, similar patterns of displacement of [³H]5–HT were seen in both tissues, with either spiperone or LSD as the unlabeled ligand. This high-affinity binding appears to be to multiple sites, since displacement studies using 2 nM [³H]5–HT result in Hill coefficients less than unity for spiperone, LSD, and quipazine [Hill coefficients (n_H): 0.44, 0.39, 0.40, respectively]. These sites apparently have an equal affinity for [³H]5-HT, since unlabeled 5-HT did not discriminate between them. Thus, the high-affinity [³H]5-HT binding in the spinal cord may be analogous to that observed in the frontal cortex, where two populations of sites have previously been described (5-HT_IA, 5-HT_IB). In addition to the multiple high-affinity spinal cord binding sites, a low-affinity [³H]5-HT binding component was also identified. A curvilinear Scatchard plot results from saturation studies using [³H]5-HT (0.5–100 nM) in the spinal cord. The plot can be resolved into sites having apparent dissociation constants of 1.4 nM and 57.8 nM for the high-and low-affinity components, respectively. Additional support for a change in affinity characteristics at higher radioligand concentrations comes from the displacement of 30 nM [³H]5-HT by the unlabeled ligand. A nonparallel shift in the dissociation curve was seen, resulting in a Hill coefficient less than unity (0.32). None of the specifically bound [³H]5-HT in the spinal cord is associated with the 5-HT uptake carrier, since fluoxetine, an inhibitor of 5-HT uptake, does not alter binding characteristics. In addition, a 5-HT binding site analogous to the site designated 5-HT, was not apparent in the spinal cord. Ketanse-rin and cyproheptadine, drugs that are highly selective for 5-HT, sites, did not displace [³H]5-HT from spinal tissue, and [³H]spiperone, a radioligand that binds with high affinity to 5-HT₂ sites, did not exhibit saturable binding in the tissue. Thus, the 5-HT₂ binding site reported in other regions of the central nervous system, and the serotonin uptake carrier do not appear to contribute to the multiple binding sites demonstrated in the spinal cord.     

Amino acid and monoamine transport in primary astroglial cultures from defined brain regions

The uptake ofl-[³H]glutamate,l-[³H]aspartate, -[³H]aminobutric acid (GABA), [³H]dopamine,dl-[³H]norepinephrine and [³H]5-hydroxytryptamine (5-HT) was studied in astrocytes cultured from the cerebral cortex, striatum and brain stem of newborn rat and grown for 2 weeks in primary cultures. The astrocytes exhibited a high-affinityl-glutamate uptake withK _m values ranging from 11 to 110 M.V _max values were 4.5 in cerebral cortex, 39.1 in striatum, and 0.4 in brain stem, nmol per mg cell protein per min. There was a less prominent high-affinity uptake ofl-aspartate withK _m values from 88 to 187 M.V _max values were 7.4 in cerebral cortex, 37.1 in striatum, and 3.1 in brain stem, nmol per mg cell protein per min. The high-affinity GABA uptake exhibitedK _m values ranging from 5 to 17 M andV _max values were 0.01 for cerebral cortex, 0.04 for striatum, and 0.1 for brain stem, nmol per mg cell protein per min. No high-affinity, high-capacity uptake was found for the monoamines. The results demonstrate a heterogeneity among the astroglial cells cultivated from the different brain regions concerning the uptake capacity of amino acid neurotransmitters. Furthermore, amino acid transmitters and monoamines are taken up by the cells in different ways.     

On the uptake and storage of 5-hydroxytryptamine, 5-hydroxytryptophan and catecholamines by adrenal chromaffin cells and nerve endings

Summary Light-microscopic autoradiographs of the adrenal medulla at various intervals after the intravenous injection of [³H] 5-HTP, [³H] 5-HT, [³H] noradrenaline and [³H] adrenaline have been studied. The distribution of silver grains following [³H] 5-HTP uptake was found to be uniform over each of the two main cell populations, adrenaline-storing (A) cells and noradrenaline-storing (NA) cells in the adrenal medulla, but A cells were twice as active as NA cells in incorporating the isotope, a situation very similar to that found after [³H] dopa uptake. 5-HT administration resulted in a pattern resembling the distribution of [³H] noradrenaline uptake, with A cells being 4 or 5 times more active than NA cells and a gradient of activity from the periphery of the medulla inwards. However, the time-course for the loss of radioactivity was not the same for both amines: levels of 5-HT activity were not significantly reduced after one week whereas the degree of [³H] noradrenaline labelling after one week was less than 10% of that at one hour. Thus 5-HT may be bound to sites in the adrenal medulla normally occupied by noradrenaline but it would appear that the release mechanism is different. There was no evidence of 5-HT uptake by adrenal nerve endings.     

Heterogeneity,position, and functional capability of the macrophages in Peyer's patches

Summary Radioenzymatic assays and light microscope radioautographic studies performed on photophores of Porichthys notatus demonstrated (1) significant amounts of catecholamines (dopamine, noradrenaline, adrenaline) and 5-hydroxytryptamine (serotonin) in these organs; (2) selective uptake and storage of [³H]noradrenaline ([³H]NA) by axon terminals innervating the photocytes, and (3) strong accumulation of [³H]5-hydroxytryptamine ([³H]5-HT) within the photocytes. Uptake and storage of [³H]NA in the nerve fibers were seemingly unaffected by the addition of ten-fold molar concentrations of unlabelled serotonin. Accumulation of [³H]5-HT by the photocytes was dose-dependent and diminished markedly in the presence of ten-fold molar concentrations of non-radioactive noradrenaline. Neither neuronal uptake of [³H]5-HT or [³H]A, nor photocytic accumulation of [³H]A were detectable under the conditions of the present experiments. This information should provide a framework for further investigations of the regulation of photophore luminescence by the biogenic amines.Supported by grants from the National Research Council and Medical Research Council of CanadaJacques de Champlain and Lise Farley provided facilities and expertise with the radioenzymatic techniques. The technical assistance of Sylvia Garcia and Marie-Hélène Parizeau was also appreciated     

Neurotransmitter uptake in various brain regions of chronically morphinized rats

Rats were long-term morphine-intoxicated by a fluid-diet model ensuring an equal nutrient intake in morphinized and control rats. Uptake of neurotransmitters and D-ala²-met⁵-enkephalinamide (Enk) was studied in the particulate fractions (obtained at 10,000g) from well defined brain regions of long-term intoxicated and morphine withdrawn rats. In control animals the accumulation of [³H]glutamate and [³H]-aminobutyric acid (GABA) reached the highest tissue/medium (T/M) ratio values, 30–120, in the regions studied while monoamine T/M values were 2–10. No active uptake of [³H]Enk could be demonstrated. Striatum showed the most evident modifications in neurotransmitter uptake. In this region the equilibrium T/M ratio for [³H]glutamate and [³H]GABA was significantly lower in intoxicated rats versus controls. Moreover, the T/M ratio for [³H]5-hydroxytryptamine (5-HT) was lower, while that for [³H]dopamine (DA) was higher in abstinent rats in comparison with the controls. [³H]glutamate and [³H]GABA uptakes were also significantly lower, respectively, in frontal cortex, hippocampus and brain stem in intoxicated rats, while [³H]5-HT uptake was significantly lower in hypothalamus after morphine withdrawal. The possible involvement of the endogenous opioid system in the etiology of the alterations is discussed.     

Functional Identification and Molecular Cloning of a Human Brain Vesicle Monoamine Transporter

A vesicle monoamine transporter was functionally identified, molecularly cloned, and characterized from a human substantia nigra cDNA library. The ATP-dependent transport of 5-[³H]hydroxytryptamine ([³H]5-HT) by digitonin-permeabilized fibroblasts expressing the vesicle monoamine/H^± antiporter in culture exhibited a K_m of 0.55 μM. Reserpine and tetrabenazine, inhibitors of two monoamine binding sites, effectively blocked [³H]5-HT accumulation with K₁ values of 34 and 78 nM, respectively. Pretreatment of cells with as little as 10 nM reserpine in the presence of ATP abolished uptake. The rank order for substrate inhibition of [³H]5-HT uptake for both the previously reported rat vMAT1 and the human transporter clone followed the order 5-HT > dopamine > epinephrine > norepinephrine > 1 -methyl-4-phen- ylpyridinium > 2-phenylethylamine > histamine. The virtually identical transport characteristics of rvMATI and hvMAT1 confirm the relevance of neuropharmacological studies of rat brain biogenic amine uptake and storage to human brain neurochemistry.     

ACTIVE UPTAKE OF [3H]5-HT BY SYNAPTIC VESICLES FROM RAT BRAIN

The question of whether synaptic vesicles accumulate [³H]5-HT by an active process was investigated in a mixed population of vesiclcs from whole rat brain. The temperature dependence and the effect of metabolic inhibitors were studied in synaptosomal suspensions and vesicular fractions. Arrhenius plots for synaptosomes differed from those for vesicles as did the temperature coefficients for these two fractions. For synaptosomes the Q₁₀ was 7 and for vesicles 1.6. However, if ATP was added to the incubation, the temperature dependence of vesicular amine accumulation became manifest; the Arrhenius plot resembled that of synaptosomes and the Q₁₀ was greater than 20 indicating strong temperature dependence. In the presence of ATP, vesicular uptake was stimulated approx 8-fold. Ouabain, dinitrophenol and NEM inhibited synaptosomal uptake but failed to affect [³H]5-HT accumulation by vesicles in the absence of ATP. When ATP was added, vesicular uptake was also blocked by NEM but was unaffected by either ouabain or DNP. Total observed uptake consisted of two components, one ATP-dependent and one nonsaturable and ATP-independent. The active process had a K_m= 1.25 × 10^?7 M and could be completely blocked by either 10^?3 M or 10^?7 M-reserpine. Active vesicular [³H]5-HT uptake was magnesium dependent and was inhibited by sodium and potassium. Cation effects on uptake were specific and could not be accounted for by either changes in osmotic pressure or ionic strength. It was concluded that synaptic vesicles from whole rat brain accumulate [³H]5-HT by an active process.     

Release of serotonin from nerve endings byl-5-hydroxytryptophan,α-methyl-m-tyramine,and elevated potassium ions

The release of [³H]5-hydroxytryptamine ([³H]5-HT) byl-5-hydroxytryptophan (L-5-HTP),-methyl-m-tyramine (-MMTA), and elevated levels of K⁺ was studied using crude synaptosomal preparations (P₂) isolated from the telencephalon of the rat and pigeon. Studies were conducted in vitro in the presence of either 2×10^–5 M tranylcypromine, which inhibited the MAO activity of both the extrasynaptosomal mitochondria and the mitochondria contained within the nerve endings (intrasynaptosomal mitochondria), or 2×10^–5 M nialamide, which inhibited the MAO activity of the extrasynaptosomal mitochondria under the experimental conditions used. In the P₂ fraction isolated from the rat, either 55 mM K⁺, 0.10 mMl-5-HTP, or 0.03 mM-MMTA significantly increased the release of [³H]5-HT above control levels, regardless of which MAO inhibitor was present in the medium. In the presence of tranylcypromine, this increased release by 55 mM K⁺ or 0.10 mMl-5-HTP was partially suppressed if Ca²⁺ was omitted from the medium. In the presence of nialamide, the release by 55 mM K⁺ was completely prevented if Ca²⁺ was omitted; the release byl-5-HTP was only partially affected. The release of [³H]5-HT by-MMTA did not appear to be markedly affected by removal of Ca²⁺, regardless of which MAO inhibitor was present. Very similar data were obtained in the presence of nialamide using the P₂ fraction isolated from the telencephalon of the pigeon, with the exception that 0.10 mMl-5-HTP caused an increase in the release of [³H]5-HIAA (which was not calcium-dependent) instead of [³H]5-HT. The data are discussed in     

Effects ofp-chlorophenylalanine on the metabolism of serotonin from 5-hydroxytryptophan

The effects ofD,L--chlorophenylalanine methyl ester (PCPA-methyl ester) and two of its metabolites, 2-(-chlorophenyl)-ethylamine (PCPEA) and -chlorophenylacetic acid (PCPAA), on the metabolism of serotonin (5-HT) fromD,L-5-hydroxytryptophan (5-HTP) ware studied in vitro and in vivo using the telencephalon and brainstem of the rat. For in vivo studies and some in vitro experiments, rats were injected with either 100 mg/kg PCPA-methyl ester or saline alone on days 1, 2, and 3, and were killed on day 15. When the in vivo metabolism of 5-HT was to be studied, the saline group and the PCPA group of animals were injected with 75 g/kg [³H]D,L-5-HTP 20 min before sacrificing. With respect to the values found for the saline-injected animals, the specific activity (S.A.; dpm/nmol) of 5-HIAA was significantly greater in the telencephanol and brainstem of the animals injected with PCPA-methyl ester. The S.A. of 5-HTP was the same in both groups; the S.A. of 5-HT was lower in the telencephalon of the PCPA group than in the saline group; in the brainstem, there was no difference. In both the saline- and PCPA-injected animals, the S.A. of 5-HIAA was greater than the S.A. of 5-HT. There was no difference between the saline- and PCPA-injected animals with regard to: (1)L-5-HTP decarboxylase activity; (2)L-5-HTP-induced release of [³H]5-HT in vitro from crude nerve ending fractions (P₂); or (3) in vitro uptake of [³H]D,L-5-HTP and its conversion to [³H]5-HT using the P₂ fraction. In vitro studies demonstrated that the PCPEA could directly cause a large increase in the release of [³H]5-HT from the P₂ fraction, whereas PCPA and PCPAA had little or no apparent effect. The data were interpreted to suggest that in the telencephalon of the animals treated with PCPA-methyl ester, there was a higher turnover of 5-HT than was found in the saline-treated group.     

[3H]Acetylcholine and [3H]5-hydroxytryptamine release from rat midbrain slices and the effects of calcium and phenobarbital

The K-stimulated release of [³H]ACh from rat midbrain slices prelabeled by incubation with [³H]choline was dependent on extracellular Ca. Phenobarbital inhibited the K-stimulated [³H]ACh release and the IC₅₀ was equal to that found for K-stimulated endogenous ACh release. These results support the suggestion that barbiturates primarily inhibit the Ca-dependent stimulated release of ACh and affect ACh synthesis only indirectly. K-Stimulated release of [³H]5-HT was also inhibited by removing Ca from the medium or by adding phenobarbital which further supports the effects of barbiturates on the depolarization-induced release process. Fluoxetine, an inhibitor of 5-HT uptake, increased the amount of [³H]5-HT found in the medium but did not fully block the uptake of [³H]5-HT in this slice preparation.     

Serotonin Regulation of Serotonin Uptake in RN46A Cells

1. Aim: The role of the serotonin transporter (SERT) is to remove serotonin (5-HT) from the synaptic space. In vitro studies have shown that 5-HT uptake via SERT is influenced by the availability of its substrate, 5-HT. We used RN46A cells, a line that expresses SERT, to investigate 5-HT regulation of 5-HT uptake and the intracellular signaling pathways involved. RN46A cells also express mRNAs for 5-HT receptors (5-HT_1A, 5-HT_1B, 5-HT_2A, and 5-HT_2C) and as cAMP and intracellular Ca²⁺ are modulated by different 5-HT receptors, we studied both pathways.2. Methods: 5-HT uptake was determined as imipramine-inhibitable uptake of [³H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca²⁺ changes were determined using the ratiometric method of intracellular Ca²⁺ imaging.3. Results: For uptake experiments, cells were kept for 30 min either with or without 1 μM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [³H]5-HT uptake. The absence of 5-HT for 15 min failed to induce any changes in intracellular cAMP levels. Removal of 5-HT from the medium did not change intracellular Ca²⁺ levels either; however, adding 1 μM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca²⁺ levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca²⁺ levels.4. Conclusions: We have shown that in RN46A cells, that endogenously express SERT and mRNAs for several 5-HT receptors, changes in 5-HT levels influence 5-HT uptake rate as well as induce changes in intracellular Ca²⁺ levels. This suggests that 5-HT may utilize intracellular Ca²⁺ to regulate 5-HT uptake.     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

Effects of a Chronic Lithium Treatment on Cortical Serotonin Uptake Sites and 5-HT1A Receptors

The objectives of this study were to characterize the effects of a chronic lithium (Li⁺) treatment on serotonin (5-HT) uptake sites and on 5-HT_1A receptors, and to determine the eventual reversibility of the treatment. The experiments were carried out with membranes from rat cerebral cortex using 8-hydroxy-2-(propylamino)tetralin, or [³H]8-OH-DPAT, and [³H]citalopram to label 5-HT_1A receptors and 5-HT uptake sites, respectively. Endogenous levels of 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) were measured by high-performance liquid chromatography in the cingulate cortex. The saturation curves with [³H]8-OH-DPAT were always best fitted a two-site model. After a treatment with Li⁺ for 28 days, no alterations in the binding parameters of [³H]8-OH-DPAT to the high- and low-affinity binding sites could be documented. However, competition curves with 5-HT to inhibit [³H]8-OH-DPAT binding revealed a decreased proportion of sites with high affinity for the agonist, together with an increased density of sites with low affinity for 5-HT, suggesting an alteration in the coupling efficacy between 5-HT_1A receptors and their transduction systems. Saturation studies with [³H]citalopram showed an increase (>40%) in the density of 5-HT uptake sites after chronic Li⁺, suggesting a more efficient 5-HT uptake process for the treated animals, in accord with clinical observations. Although 5-HT contents in cingulate cortex remained unchanged after the treatment, 5-HIAA levels decreased (>30%), leading to a diminished (almost 50%) 5-HT turnover; and also reflecting a more efficient uptake in the treated rats, so that less 5-HT could be degraded by extracellular monoamine oxidase. All the effects revealed by [³H]8-OH-DPAT and [³H]citalopram were reversed following a recovery period of two days without Li⁺. Since symptoms of bipolar affective disorders may reappear if the chronic Li⁺ treatment is interrupted, the reversibility of the observed effects further supports the importance of central 5-HT synaptic transmission in the pathophysiology and treatment of human affective disorders.     

N-Methyl-d-Aspartic Acid/Glycine Interactions on the Control of 5-Hydroxytryptamine Release in Raphe Primary Cultures

Abstract: Glutamic acid and glycine were quantified in cells and medium of cultured rostral rhombencephalic neurons derived from fetal rats. In the presence of 1 mM Mg²⁺, NMDA (50 μM) significantly stimulated (by 69%) release of newly synthesized 5-[³H]hydroxytryptamine ([³H]5-HT). d -2-Amino-5-phosphonopentanoate (AP-5; 50 μM) blocked the stimulatory effect of NMDA. AP-5 by itself inhibited [³H]5-HT release (by 25%), suggesting a tonic control of 5-HT by glutamate. In the absence of Mg²⁺, basal [³H]5-HT release was 60% higher as compared with release with Mg²⁺. AP-5 blocked the increased [³H]5-HT release observed without Mg²⁺, suggesting that this effect was due to the stimulation of NMDA receptors by endogenous glutamate. Glycine (100 μM) inhibited [³H]5-HT release in the absence of Mg²⁺. Strychnine (50 μM) blocked the inhibitory effect of glycine, indicating an action through strychnine-sensitive inhibitory glycine receptors. The [³H]5-HT release stimulated by NMDA was unaffected by glycine. In contrast, when tested in the presence of strychnine, glycine increased NMDA-evoked [³H]5-HT release (by 22%), and this effect was prevented by a selective antagonist of the NMDA-associated glycine receptor, 7-chlorokynurenate (100 μM). 7-Chlorokynuren-ate by itself induced a drastic decrease in [³H]5-HT release, indicating that under basal conditions these sites were stimulated by endogenous glycine. These results indicate that NMDA stimulated [³H]5-HT release in both the presence or absence of Mg²⁺. Use of selective antagonists allowed differentiation of a strychnine-sensitive glycine response (inhibition of [³H]5-HT release) from a 7-chlorokynurenate-sensitive response (potentiation of NMDA-evoked [³H]5-HT release).     

Adult development of the hippocampal-serotonin system of C57BL/6N mice; analysis of high-affinity uptake of 3H-5HT in slices and synaptosomes

The saturable and specific high-affinity uptake of [³H]serotonin ([³H]5HT) (5 × 10^?8 M) was studied in slices from the hippocampus, parietal cortex, septum-preoptic area, and hypothalamus of male 2, 6, 12 and 24–32 month old C57BL/6N mice. Hippocampal [³H]5-HT uptake showed a significant biphasic relationship to age, with lower values in the 2 and 24–32 month old mice compared to 6 month old mice. No significant age effects were seen in the other regions, or in [³H]norepinephrine high-affinity uptake in the hippocampus.Studies of the high-affinity uptake mechanism in synaptosomal preparations were made in a subgroup of 12 and 24 month old mice. A micro-assay using a tissue-harvester measured high-affinity uptake on 8–30 μl of the P₂ suspension (crude-synaptosomal preparation). The high-affinity uptake was linear for 4 min at 37°C and inhibited in both the adult and aged tissue by 10^?5 M cold 5-HT (83 and 78% respectively), 10^?5 M fluoxetine (85 and 82% respectively) and 10^?3 M NaCN (57 and 57% respectively). Kinetic analysis of the [³H]5HT high-affinity uptake in the hippocampus (3 min, 37°C) revealed the same apparent K_m for serotonin at both ages (6.7 x 10^?8 M), but a 44% decrease in V_max in the aged hippocampal synaptosomal high-affinity uptake compared to adults (120 vs 215 pmol of 5-HT/g-tissue/3 min).These results are discussed in relationship to the reported age effects on the intrinsic neurons of the hippocampus.     

FEEDBACK REGULATION OF 5-HT SYNTHESIS IN RAT STRIATAL SLICES

The effects of changes in intraneuronal levels of 5-HT induced by monoamine oxidase inhibitors (MAOI) given in vivo or exogenous 5-HT added in vitro on 5-HT synthesis in striatal slices of the rat have been examined. The synthesis of 5-HT was estimated by the measurement of the total formation of [³H]5-HT and [³H]5-hydroxyindole acetic acid from [³H]tryptophan and by calculation of the conversion index (CI) of tryptophan into 5-HT. The small formation of [³H]tryptamine and [³H]indole acetic acid from [³H]tryptophan was taken into account in the estimation of 5-HT synthesis. Both MAOI pretreament (180 min) and 5-HT (2·8 μM) inhibited synthesis. The latter effect persisted in catecholamine depleted tissues and was related to intraneuronal changes in 5-HT levels, since it could be prevented by chlorimipramine. The inhibition of 5-HT synthesis was related to the decreased conversion of tryptophan into 5-hydroxytryptophan and could not be prevented by p-chloro-phenylalanine pretreatment which depleted 5-HT levels or by dibutyryl cyclic AMP which normally stimulated 5-HT synthesis. Tryptophan uptake in slices was not affected by exogenous 5-HT. The various mechanisms possibly involved in the end product regulation process of 5-HT synthesis are discussed.