Characterization of the effects of serotonin on the release of [3H]dopamine from rat nucleus accumbens and striatal slices

The effect of serotonin agonists on the depolarization (K⁺)-induced, calcium-dependent, release of [³H]dopamine (DA) from rat nucleus accumbens and striatal slices was investigated. Serotonin enhanced basal³H overflow and reduced K⁺-induced release of [³H]DA from nucleus accumbens slices. The effect of serotonin on basal³H overflow was not altered by the serotonin antagonist, methysergide, or the serotonin re-uptake blocker, chlorimipramine, but was reversed by the DA re-uptake carrier inhibitors nomifensine and benztropine. With the effect on basal overflow blocked, serotonin did not modulate K⁺-induced release of [³H]DA in the nucleus accumbens or striatum. The serotonin agonists, quipazine (in the presence of nomifensine) and 5-methoxytryptamine, did not significantly affect K⁺-induced release of [³H]DA in the nucleus accumbens. This study does not support suggestions that serotonin receptors inhibit the depolarization-induced release of dopamine in the nucleus accumbens or striatum of the rat brain. The present results do not preclude the possibility that serotonin may affect the mesolimbic reward system at a site which is post-synaptic to dopaminergic terminals in the nucleus accumbens.     

Photoaffinity labeling of serotonin-binding proteins

A photosensitive arylazide derivative of serotonin (nitroaryl-azidophenyl serotonin, NAP-serotonin) has been synthesized for use in studying the biochemical nature of serotonin binding sites. [³H]-NAP-serotonin possesses a similar ability to bind to the crude membranes of rat brains does [³H]-serotonin and therefore seems suitable for use as a photoaffinity labeling probe for serotonin binding sites. Upon irradiation with ultraviolet light, [³H]-NAP-serotonin covalently attaches to protein components of the brain homogenate. Several distinct radioactively labeled proteins have been separated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis. Their apparent molecular weights were 80,000, 49,000, and 38,000 (±5%). When 1 μM of unlabeled serotonin or d-lysergic acid diethylamide (d-LSD) was added prior to photolysis, the incorporation of [³H]-NAP-serotonin into these proteins was inhibited significantly. No inhibitory effect was observed when dopamine was used. These observations suggest that the photoaffinity labeled proteins are specific for serotonin binding.     

Heartwood extract of Rhus verniciflua Stokes and its active constituent fisetin attenuate vasoconstriction through calcium-dependent mechanism in rat aorta

Rhus verniciflua Stokes (RVS) exert cardiovascular protective activity by promoting blood circulation, but its active ingredients and underlying mechanism have yet to be identified. This study investigated the vascular effects of RVS, focusing on vasoconstriction and smooth muscle Ca²⁺ signaling. RVS heartwood extract attenuated contraction of aortic rings induced by the vasoconstrictors serotonin and phenylephrine, and inhibited the Ca²⁺ signaling evoked by serotonin in vascular smooth muscle cells. Subsequent activity-guided fractionation identified fisetin as an active constituent exerting a Ca²⁺ inhibitory effect. Fisetin could inhibit major Ca²⁺ mobilization pathways including extracellular Ca²⁺ influx mediated by the L-type voltage-gated Ca²⁺ channel, Ca²⁺ release from the intracellular store and store-operated Ca²⁺ entry. In accordance with Ca²⁺ inhibitory effect, fisetin attenuated vasoconstriction by serotonin and phenylephrine. These results suggest that the anticontractile effect, which is presumably mediated by inhibition of Ca²⁺ signaling, may contribute to the improvement of blood circulation by RVS.     

In vivo [3H]ketanserin binding: effect of modifying synaptic serotonin levels

Most antidepressant therapies aim to increase the synaptic concentration of one or more of the monoamines. Synaptic monoamine levels are, however, extremely difficult to measure. In order to estimate synaptic levels of serotonin, an indirect method has been used. Since [³Hjketanserin is an antagonist at the 5HT₂ serotonin receptor, one would expect its in vivo binding to be inhibited by increased levels of synaptic serotonin. This hypothesis was tested in mice by measuring the effect of compounds which are considered to raise the synaptic concentration of serotonin. Directly acting agents such as quipazine, methysergide and mianserin inhibited [³H]ketanserin binding in vitro and in vivo. On the other hand, indirect agonists such as the monoamine oxidase inhibitors, pargyline and niamide, the serotonin uptake blockers, paroxetine and midalcipran, the serotonin releasers, p-chloroamphetamine and H75/12 and the serotonin precursor, 5-hydroxytryptophan had no effect on [³H]ketanserin binding in vivo. This was in spite of the fact that at doses used a very marked serotonin-induced behaviour was observed. In view of its insensitivity to changes in synaptic concentrations of serotonin, it is possible that the sites labelled in vivo by [³H]ketanserin are not innervated by the serotonin nerve terminals through which these indirect serotonin agonists act.     

A Highlights from MBoC Selection: Calcineurin-dependent cofilin activation and increased retrograde actin flow drive 5-HT–dependent neurite outgrowth in Aplysia bag cell neurons

Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca²⁺; however, mechanistic roles for Ca²⁺ in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) → inositol trisphosphate → Ca²⁺ → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca²⁺ release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT–dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.     

Serotonin Modulation of Low-Affinity Ouabain Binding in Rat Brain Determined by Quantitative Autoradiography

Previous results showed that Na⁺/K⁺-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na⁺/K⁺-ATPase activity and [³H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which isoform is involved in the Na⁺/K⁺-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [³H]ouabain binding to rat brain slices was employed at different [³H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [³H]ouabain binding was sensitive to serotonin 10^–3 M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [³H]ouabain concentrations, strongly suggests the participation of the 2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K⁺ handling by the glial 2 isoform of the pump.     

DETECTION OF A SOLUBLE SEROTONIN-BINDING PROTEIN IN THE MAMMALIAN MYENTERIC PLEXUS AND OTHER PERIPHERAL SITES OF SEROTONIN STORAGE

Groups of neurons intrinsic to the mammalian myenteric plexus have been shown to have both tryptophan hydroxylase and a specific uptake mechanism for serotonin. They are probably serotonergic. A soluble protein with a high binding affinity for serotonin, similar to a protein previously found in rat brain by TAMIR & HUANG (1974), has now been found in the myenteric plexus of both rabbit and guinea pig. Partial purification of the protein from the rabbit's myenteric plexus by ammonium sulfate fractionation increased the ratio of specific to nonspecific serotonin binding almost 3-fold. Two dissociation constants for serotonin binding were obtained by equilibrium dialysis: 6.7 × 10^?10 M and 4.8 × 10^?7 M. The protein was similar to the soluble serotonin-binding protein of CNS: the indole derivatives 5, 6- and 5, 7-dihydroxytryptamine, and 6-hydroxytryptamine inhibited serotonin binding by 50% at 10^?7 M; norepinephrine was a poor inhibitor of serotonin binding; most of the serotonin-protein complex had a very high molecular weight and did not penetrate a 6.5% acrylamide gel. The appearance of the serotonin binding protein during development of the intestine in fetal rabbit correlates closely with the development of a serotonin uptake mechanism by nerves of this tissue and precedes the ingrowth of the adrenergic innervation. In-vitro administration of 6-hydroxydopamine to adult animals has no effect on the binding capacity for serotonin. Binding activity in denervated preparations is only 1/5 that of innervated tissue. It is concluded that the serotonin-binding protein, which has been found associated with serotonergic pathways in the CNS, is found associated with serotonergic neurons in the periphery as well. Since a similar serotonin-binding protein is also found in sheep thyroid, which stores but does not take up serotonin, the protein may be a component of the serotonin storage mechanism.     

2-Nitroimipramine: A selective irreversible inhibitor of [3H] serotonin uptake and [3H] imipramine binding in platelets

The effect(s) of a new imipramine analogue, 2-nitroimipramine, on high affinity [³H] imipramine binding and [³H] serotonin uptake in human platelets were studied. 2-Nitroimipramine was found not only to be a very potent inhibitor of [³H] imipramine binding and [³H] serotonin uptake but was found to irreversibly inhibit binding and uptake simultaneously. This finding supports previous observations from our laboratory and others that high affinity imipramine binding labels serotonin uptake or transport sites. 2-Nitroimipramine should prove an important tool for subsequent studies of the molecular mechanism(s) involved in the transport of serotonin and the binding of imipramine to platelet and brain membranes.     

Biogenic amines in cultured neuroblastoma and astrocytoma cells

The presence of biogenic amines in cultured cells of mouse neuroblastoma C-1300 (clone NB-2a) was suggested by fluorescence-microscope histochemistry. Incubation in media containing L-[¹⁴C]tyrosine and L-[¹⁴C]tryptophan for 24 h, followed by high-voltage electrophoresis, radiochromatogram scanning, and scintillation counting, confirmed the presence of [¹⁴C]dopamine, [¹⁴C]norepinephrine, [¹⁴C]epinephrine, [¹⁴C]serotonin, [¹⁴C]tyramine, and [¹⁴C]octopamine. Dopamine, norepinephrine, epinephrine, and serotonin were demonstrated spectrophotofluorometrically in concentrations, expressed as micrograms amine per milligram protein, of 1.19, 0.027, 0.038, and 0.148, respectively, for cells in a stationary growth phase. Fluorescence-microscope histochemistry also suggested the presence of biogenic amines in cultured astrocytoma cells (cell line C6). Spectrophotofluorometric assay of cells in a stationary growth phase demonstrated intracellular dopamine, norepinephrine, epinephrine, and serotonin in concentrations significantly lower than those of neuroblastoma cells.     

Guanine Nucleotides Modulate Cortical S2 Serotonin Receptors

Abstract

Many radiolabelled receptors coupled to intracellular adenylate cyclase activity have been found to be modulated by physiological modulators such as GTP (guanosine triphosphate) and Gpp(NH)p (guanosine-imido-diphosphate). In particular, the apparent affinity of agonists competing for the binding of ³H-antagonist-labelled receptors is reduced in the presence of GTP and Gpp(NH)p. We report herein the agonist-specific effects of GTP and Gpp(NH)p on rat brain cortical S² serotonin receptors. The agonists serotonin, 5-methoxytryptamine, bufotenine, and tryptamine display threefold lower affinities for S₂ serotonin receptors in the presence of 10^-4M GTP or Gpp(NH)p than in the absence of the nucleotides. The antagonists spiperone, cinanserin, cyproheptadine and methysergide are unaffected by the guanine nucleotides. The Hill coefficients of the agonists increase from between 0.70–0.80 to 0.90–1.00 due to guanine nucleotides. ATP, ADP, and GDP have little or no effect. This pattern of guanine nucleotide effects has been found with receptors which are modulated by a guanine nucleotide regulatory protein and may indicate that the S₂ serotonin receptor may be coupled to intracellular adenylate cyclase activity.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.     

Effect of ibogaine on serotonergic and dopaminergic interactions in striatum from mice and rats

The effect of ibogaine (Endabuse, NIH 10567) on serotonin uptake and release, and on serotonergic modulation of dopamine release, was measured in striatal tissue from rats and mice. Two hours after treatment in vivo with ibogaine (40 mg/kg i.p.), the uptake of labeled [³H]serotonin and [³H]dopamine uptake in striatal tissue was similar in the ibogaine-treated animal to that in the control. The 5HT_1B agonist CGS-12066A (10^–5 M) had no effect on stimulation-evoked tritium release from mouse or rat striatal tissue preloaded with [³H]serotonin; however, it elevated tritium efflux from striatal tissue preloaded with [³H]dopamine. This increase was not seen in mice treated with ibogaine 2 or 18 hours previously, or in rats treated 2 hours before. Dopamine autoreceptor responses were not affected by ibogaine pretreatment in either mouse or rat striatal tissue; sulpiride increased stimulation-evoked release of tritium from tissue preloaded with [³H]dopamine. The long-lasting effect of ibogaine on serotonergic functioning, in particular, its blocking of the 5HT_1B agonist-mediated increase in dopamine efflux, may have significance in the mediation of its anti-addictive properties.Special issue dedicated to Dr. Sidney Ochs.     

Serotonin has both excitatory and inhibitory modulatory effects on feeding muscles in Aplysia

Serotonin (10^?8M) produced opposite long-lasting (up to 10 min) effects on acetylcholine-elicited contractions of different buccal mass muscles of Aplysia. Contractions of the dorsal extrinsic muscle and accessory radula closer muscle were enhanced by serotonin; whereas contractions of the ventral extrinsic muscles were inhibited by serotonin. The effect of higher concentrations of serotonin on dorsal and ventral extrinsic muscles was in the same direction as at 10^?8M but was greater in both magnitude and duration. The phase of feeding-protraction or retraction—during which a muscle is active—is not correlated with the direction of modulation produced by serotonin.     

Glycosaminoglycan mimetics trigger IP3-dependent intracellular calcium release in myoblasts

Glycosaminoglycans (GAG) are sulfated polysaccharides that play an important role in regulating cell functions. GAG mimetics called RGTAs (for ReGeneraTing Agents) have been shown to stimulate tissue repair. In particular they accelerate myogenesis, in part via their heparin-mimetic property towards growth factors. RGTAs also increase activity of calcium-dependent intracellular protease suggesting an effect on calcium cellular homeostasis. This effect was presently investigated on myoblasts in vitro using one member of the RGTA family molecule named OTR4120. We have shown that OTR4120 or heparin induced transient increases of intracellular calcium concentration ([Ca²⁺]i) in pre-fusing myoblasts from both mouse SolD7 cell line and rat skeletal muscle satellite cells grown in primary culture by mobilising sarcoplasmic reticulum store. This [Ca²⁺]i was not mediated by ryanodine receptors but instead resulted from stimulation of the Inositol-3 phosphate-phospholipase C activation pathway. OTR4120-induced calcium transient was not mediated through an ATP, nor a tyrosine kinase, nor an acetylcholine receptor but principally through serotonin 5-HT2A receptor. This original finding shows that the GAG mimetic can induce calcium signal through serotonin receptors and the IP3 pathway may be relevant to its ability to favour myoblast differentiation. It supports a novel and unexpected function of GAGs in the regulation of calcium homeostasis.     

Differential binding of 3H-imipramine and 3H-mianserin in rat cerebral cortex

Drug competition profiles, effect of raphé lesion, and sodium dependency of the binding of two antidepressant drugs ³H-imipramine and ³H-mianserin to rat cerebral cortex homogenate were compared to examine whether the drugs bound to a common “antidepressant receptor.” Of the neurotransmitters tested, only serotonin displaced binding of both ³H-imipramine and ³H-mianserin. ³H-mianserin binding was potently displaced by serotonin S₂ antagonists and exhibited a profile similar to that of ³H-spiperone binding. In the presence of the serotonin S₂ antagonist spiperone, antihistamines (H₁) potently displaced ³H-mianserin binding. ³H-Imipramine binding was displaced potently by serotonin uptake inhibitors. The order of potency of serotonergic drugs in displacing ³H-imipramine binding was not similar to their order in displacing ³H-spiperone or ³H-serotonin binding. Prior midbrain raphé lesions greatly decreased the binding of ³H-imipramine but did not alter binding of ³H-mianserin. Binding of ³H-imipramine but not ³H-mianserin was sodium dependent. These results show that ³H-imipramine and ³H-mianserin bind to different receptors. ³H-Imipramine binds to a presynaptic serotonin receptor which is probably related to a serotonin uptake recognition site, the binding of which is sodium dependent. ³H-Mianserin binds to postsynaptic receptors, possibly both serotonin S₂ and histamine H₁ receptors, the binding of which is sodium independent.     

Possible Participation of Serotonin in the Peripheral Link of the Defensive Reflex in the Mollusc Lymnaea stagnalis

The contractions of the dorsal longitudinal muscle of the mollusc Lymnaea stagnalis L., which are evoked by electric stimulation of n. cervicalis inferior were studied. It has been shown that an increase of magnesium ion concentration in saline to 10–15 mM decreases reversibly amplitude of the evoked contractions. Application of serotonin produced a dual effect: at concentrations of 2 × 10^–5–10^–6 M, it enhanced muscle contractions, whereas at concentrations above 10-5 M, on the contrary, decreased them. The inhibitory effect of the serotonin antagonist mianserin on the evoked contraction amplitude increased with elevation of its concentrations in the studied range (from 10^–5 to 10^–3 M). The enhancing effect of serotonin on muscle contractions was blocked either by previous mianserin application or its application on the background of the already acting serotonin. A participation of serotoninergic mechanisms in the control of the contractile function of the studied muscle is suggested.     

On the Nature of the Interaction Between Serotonin and Serotonin Binding Protein: Effect of Nucleotides, Ions, and Sulfhydryl Reagents

Abstract: Rat brain serotonin binding protein (SBP) was found to have essential -S-S and -SH groups. Both reduction of the disulfide bond by dithiothreitol or mercaptoethanol and modification of -SH group(s) by Ellman reagent or alkylating agents caused loss of binding capacity. In contrast, formation of a mixed disulfide bond with sodium metabisulfite did not affect the binding capacity. Serotonin in the presence of Fe²⁺ and phosphate was found to bind to either an -SH group or to a site in very close proximity. Addition of serotonin protected -SH groups from modification by Ellman reagent and from denaturation of protein upon storage. Lipids that enhance binding of serotonin to SBP also protected -SH groups from modification. Nucleotides were found to be strong inhibitors of the binding of serotonin to SBP. The inhibitory effect of nucleotides was due to their chelating properties and not to their ability to phosphorylate the protein or to bind directly to it. Inhibition by nucleotides and other chelators was reversible. Binding capacity was fully restored after removal of the chelator by molecular sieve chromatography and addition of Fe²⁺. The ionic environment had a marked effect on the binding: intracellular ions such as K⁺ were found to enhance the binding, and extracellular ions such as Na⁺ and Ca²⁺ inhibited the binding. Based on these data and our previous studies, we suggest that SBP is an intracellular protein that acts as a storage protein. Consistent with our data is formation of a complex of SBP-S-Fe-S that in a hydrophobic surrounding could bind up to four molecules of serotonin in coordination bond with Fe²⁺ and thereby reduce the osmotic pressure within a storage vesicle. Extracellular ionic conditions that favor the dissociation of the complex would free the amine to interact with its receptor or the presynaptic reuptake carrier.     

High-affinity uptake of [3H]serotonin in cultured neurones of the cockroach Periplaneta americana

Cultured central neurons from the American cockroach, Periplaneta americana, have been used to investigate the uptake of [³H]serotonin. The neurones accumulate [³H]serotonin from the extracellular medium by both a high-and a low-affinity system. The activity of the high-affinity mechanism is decreased by low temperature and metabolic poisons, and is dependent on sodium and chloride ions. Both depolarising levels of external potassium ions and veratridine decrease the high-affinity uptake system, suggesting it is influenced by the transmembrane potential. The pyrethroid insecticides, deltamethrin and permethrin, enhance the inhibitory effect of veratridine. Pyrethroid enhancement is completely blocked by tetrodotoxin, and neither pyrethroid affects the uptake system in the absence of veratridine. Avermectin B_1A is a powerful inhibitor of the high-affinity uptake system, and its effect is blocked by picrotoxin. High-affinity uptake of [³H]serotonin is inhibited by imipramine and amitriptyline; desipramine has no significant effect on this uptake. The activity of the high-affinity system is also reduced by 8-hydroxy-dipropylaminotetralin, α-methyl-serotonin, and 1-(3-chlorophenyl)piperazine. Dopamine, noradrenaline, octopamine, and the formamidine insecticides, chlordimeform and demethylchlordimerform, are moderate inhibitors of the high-affinity uptake system. The formamidine effect is not blocked by tetrodotoxin or picrotoxin.     

Does high affinity [3H] imipramine binding label serotonin reuptake sites in brain and platelet?

Recent studies have demonstrated the presence of high affinity binding sites for [³H] imipramine in membrane preparations derived from rat brain, human platelet, and human brain. Although initial reports concluded that there was no relationship between these binding sites and the reuptake sites for biogenic amines, subsequent studies in our laboratory suggested a close relationship between the high affinity imipramine binding site and the serotonin uptake or transport site (cf. ref. 9). To further establish whether these binding sites are associated with either platelet or neuronal uptake of serotonin, the relative potencies of a series of tricyclic antidepressants in inhibiting [³H] imipramine binding and serotonin uptake were determined under identical assay conditions. A close correlation between inhibition of serotonin uptake and [³H] imipramine binding was observed (r = 0.99, p < 0.001). In addition, electrolytic lesions of the midbrain raphe produced a decrease in [³H] imipramine binding in hypothalamic synaptosomes that paralleled the decrease in [³H] serotonin uptake. Finally incubation of synaptosomal membranes with 2,8-dinitroimipramine, an irreversible inhibitor of [³H] imipramine binding, produced a dose-dependent decrease in serotonin uptake, without altering the uptake of nonrepinephrine or dopamine. Taken together our results strongly suggest that high affinity binding of [³]] imipramine selectively labels serotonin uptake sites in brain and platelet.     

Decreased [3H]serotonin and [3H]spiperone binding consequent to lipid peroxidation in rat cortical membranes

The effect of lipid peroxidation on two types of serotonin binding sites was investigated. Incubation of rat cortical membranes with an ascobate-dependent peroxidizing system resulted in the formation of malonyldialdehyde and significant decreases in the specific binding of [³H]serotonin and [³H]spiperone to the treated membranes. When ascorbate concentrations were varied from 0.025 to 6.0 mM, malonyldialdehyde production increased to a maximum at 0.5 mM ascorbate and then declined. Conversely, the specific binding of [³H]serotonin and [³H]spiperone decreased to a minimum at 0.5 mM ascorbate and then increased. Regression analysis of the data revealed that the decrease in the two binding sites was linearly correlated to lipid peroxidation.