Butanol extracts from myelin fragments: identification of 5-hydroxytryptamine binding components

To investigate the nature of 5-HT binding components originated from myelin butanol extracts (i.e. proteolipids), the recombinate experiments with acidic lipids were planned. Binding assay of 14C . 5-Ht to recombinates was carried out by Sephadex lh20 column chromatography. Among several recombinate fractions, recombinate of sulphatides, phosphatidylserine and phosphatidylinositol showed the same elution profile and binding capacity for 14C . 5-HT with those of the myelin extracts. The displacement studies with other neurotransmitters revealed that the recombinate of these three acidic lipids completely regenerated the specificity of the original myelin proteolipids. Namely, tryptamine, DA and ACh inhibited the 5-HT binding but NA had no effect. A double reciprocal plot of 5-HT binding represented a multiple binding mode, not a straight line. This observation suggested that, at least, three binding components (or sites) are implicated in the binding of 14C . 5-HT to this recombinate system having 3 Kds of 2.2 x 10(-7), 4.1 x 10(-7) and 4.2 x 10(-6) m. All these observations infer that the 5-HT binding components of myelin proteolipids are mainly three acidic lipids in nature.     

Identification of 5-hydroxytryptamine binding substances from rat brain stem

Abstract— Godwin & Sneddon (1975) reported the binding of 5-hydroxy-[³H]tryptamine (5-HT) on a Sephadex LH-20 column to‘proteolipid material’extracted with n-butanol from rat brain stem. An examination of this‘proteolipid material’with TLC showed the main constituents to be cerebroside sulfate (CS), monophosphoinositide (PI), and diphosphoinositide. The elution profiles of [³H]5-HT incubated with purified CS or with a mixture of CS and PI were similar to that of the brain extract on the same column. Because the elution profile of the mixture of CS and PI was more similar to that of the brain extract, it was concluded that what was suggested to be a possible proteolipid‘5-HT receptor’was mainly two acidic lipids. The elution profile of [³H]5-HT incubated with purified PI, however, was similar to [³H]5-HT eluted alone. This suggested that either PI did not bind to 5-HT or that the PI-5-HT complex possesses different Chromatographie behavior than PI. To test this latter possibility, [¹⁴C]5-HT and [³H]PI were incubated then eluted on a Sephadex LH-20 column with a continuous gradient of increasing polarity. The gradient first eluted PI, then an apparent PI-5-HT complex, and finally 5-HT. This demonstrated that PI will bind to 5-HT on a Sephadex LH-20 column and that the PI-5-HT complex is probably more polar than PI.     

Subcellular distribution and chemical nature of the receptor for 5-hydroxytryptamine in the central nervous system

Abstract— In vitro binding experiments with 5-hydroxy[¹⁴C]tryptamine (3.3 × 10^?6 M) were carried out on subcellular fractions of the cat brain. The highest specific activity was observed in some fractions of nerve-ending membranes isolated from the hypothalamus, basal ganglia, and gray areas of the mesencephalon. The specificity of this high affinity binding was demonstrated by competition with reserpine, butanolamide of lysergic acid, and desmethylimipramine. With butanol-water extraction the [¹⁴C]5-HT was found in the butanol while the gangliosides were separated in the water phase. Several experiments with thin layer and column chromatography suggest that in the organic phase the [¹⁴C]5-HT is not bound to the lipids but to a special proteolipid. This proteolipid is different from that found in myelin and has similar chromatographic properties to that previously observed in the proteolipid which binds d-[¹⁴C]tubocurarine in nerve-ending membranes of the cerebral cortex.     

Autoreceptor-mediated inhibition of 3H-5-hydroxytryptamine release from rat brain cortex slices by analogues of 5-hydroxytryptamine

Rat brain cortex slices preincubated with ³H-5-hydroxytryptamine (³H-5-HT) were superfused with physiological salt solution containing paroxetine, an inhibitor of 5-hydroxytryptamine (5-HT) uptake. The effects of various indolethylamines on the electrically evoked tritium overflow (containing 66.3% unmetabolized ³H-5-HT) were investigated (the percentage of unmetabolized ³H-5-HT was not altered by the indolethylamines or metitepin). 6,7-Dihydroxytryptamine (6,7-DHT) did not affect the stimulation-evoked tritium overflow, whereas the latter was inhibited by the other tryptamine derivatives investigated; when the compounds were compared to each other on the basis of their inhibitory potencies the following rank order was obtained: unlabelled 5-HT > 5-methoxytryptamine > 4-HT > 6-HT > 5,6-DHT > tryptamine > 7-HT > 5,7-DHT. The inhibitory effects of these compounds were antagonized by metitepin. It is concluded that the indolethylamines inhibit the stimulation-evoked ³H-5-HT release by activating the presynaptic 5-HT autoreceptors on the 5-HT neurones of the rat brain cortex. Similarities may exist between these receptors and the postsynaptic 5-HT_l binding sites of this brain area.     

AN ASSAY PROCEDURE FOR TRYPTAMINE IN BRAIN AND SPINAL CORD USING ITS [3H]DANSYL DERIVATIVE

—The tryptamine content of rat and mouse brain and spinal cord was determined with a radiochemical derivative assay, using [³H]dansyl chloride. The amine was extracted into toluene-isoamyl alcohol, back-extracted into dilute acid, then adsorbed onto a non-ionic polystyrene resin, and dansylated in tetrahydrofuran after elution from the resin. Optimum recoveries were obtained with TCA extracts, although significant losses occurred due to surface adsorption and protein binding. The brain content of tryptamine increased after MAO inhibition and was not significantly further increased when tryptophan loading was combined with inhibition of MAO and/or tryptophan 5-hydroxylase. The tryptamine concentration of spinal cord exceeded that of brain and increased rapidly after death. Among brain regions tryptamine concentrations were greatest in hypothalamus and striatum and lowest in cerebral cortex and cerebellum.     

The binding of 5-hydroxytryptamine to acidic lipids in isobutanol.

Abstract— In order to examine the possibility that acidic lipids can account for the binding of 5-hydroxy [³H]tryptamine (5-HT) to brain tissue, the binding to six acidic lipids was studied using an isobutanol-water partition method. With the exception of the polyphosphoinositides, all the acidic lipids examined bind saturably and with high affinity. The apparent dissociation constants of 5-HT to the acidic lipids were as follows: phosphatidylserine, 0.4 μM; phosphatidic acid, 0.6μM; diphosphoinositide, 0.8 μM; cerebroside sulfate, 1.4 μM; monophosphoinositide, 1.9 μM; and triphosphoinositide, 10 μM. The high affinity of these lipids to 5-HT raises the possibility of some role for them in serotonergic activity.     

Extracellular calcium dependence of contracture and modulation by serotonin in buccal muscle E1 of Aplysia

Serotonin [5-hydroxytryptamine (5-HT)] enhances acetyl choline (ACh)-elicited contractures of Aplysia buccal muscles E1 and I5. The possible role of external calcium in regulating the magnitude of ACh contracture in the presence and absence of 5-HT was investigated. Superfusion of E1 with zero calcium medium caused ACh contractures to fail within one to two minutes. Recovery of ACh contracture upon restoring normal medium occurred within two to four minutes. In the absence of 5-HT, ACh contracture decreased proportionally to external [Ca⁺⁺] in the concentration range of 0–10 mM; however, the amount of enhancement of of ACh contracture following 5-HT treatment did not decrease with external [Ca⁺⁺] as long as [Ca⁺⁺] was above a threshold concentration that varied from preparation to preparation. For most preparations, the enhancement of ACh contracture by 5-HT was dependent on the presence of external calcium during 5-HT treatment. Calcium influx into muscles E1 and I5 increased approximately two and a half fold in the presence of 10^?6 M 5-HT. A model in which 5-HT brings about calcium “loading” of an ACh releasable intracellular storage site is discussed.     

The activation of phosphatidylinositol turnover is not directly involved in the modulation of neurotransmitter release mediated by presynaptic muscarinic receptors

In the rat cerebral cortex, the comparative effects of various muscarinic agonists on the release of [³H]dopamine ([³H]DA), [³H]acetylcholine ([³H]ACh), and [³H]5-hydroxytryptamine ([³H]5-HT) from superfused nerve endings and on phosphatidylinositol (PI) turnover were studied. Acetylcholine (ACh) was found to be the most potent among the agonists tested on all three release systems examined, and also on the activation of PI turnover. Oxotremorine and bethanechol were very weak agonists when tested as stimulators of PI turnover. However, oxotremorine was very effective as a release modulator, while bethanechol was completely ineffective. Our data suggest that the activation of PI turnover is not directly involved in the modulation of neurotransmitter release mediated by presynaptic muscarinic receptors.     

N-Acetylneuraminic acid molecules as possible serotonin binding sites.

5-Hydroxytryptamine (5-HT), but not acetylcholine, carbamylcholine or L-D-noradrenaline, binds to ox brain ganglioside micelles, to phosphatidylcholine smectic mesophases (liposomes) containing gangliosides and to the glycoprotein fetuin, through the negatively charged N-acctylneuraminic acid (NeuNAc) residues. The 5-HT binding to NeuNAc is reversible, saturable, prodeeds in a 1: 1 fashion and can be specifically blocked by 7-methyltryptamine. Thc affinity constant at equilibrium for the reaction is of the order of 10² 1. mol-¹. No special ganglioside was identified as specifically associating with the amine. A terminal NeuNAc in the gangliosides is not a prerequisite for binding, although it seems important for binding 5-HT in entire mcmbrane preparations (MARCHBANKS, 1966) or for bringing about the 5-HT induced contraction of smooth muscle cells (WOOLLCY & GOMML 1964a). It is proposed that in 5-HT target cells, NeuNAc residues, probably attached to membrane surface glycoprotein(s) are involved in thc mechanism of action of the drug.     

A Homolog of FHM2 Is Involved in Modulation of Excitatory Neurotransmission by Serotonin in C. elegans

The C. elegans eat-6 gene encodes a Na⁺, K⁺-ATPase α subunit and is a homolog of the familial hemiplegic migraine candidate gene FHM2. Migraine is the most common neurological disorder linked to serotonergic dysfunction. We sought to study the pathophysiological mechanisms of migraine and their relation to serotonin (5-HT) signaling using C. elegans as a genetic model. In C. elegans, exogenous 5-HT inhibits paralysis induced by the acetylcholinesterase inhibitor aldicarb. We found that the eat-6(ad467) mutation or RNAi of eat-6 increases aldicarb sensitivity and causes complete resistance to 5-HT treatment, indicating that EAT-6 is a component of the pathway that couples 5-HT signaling and ACh neurotransmission. While a postsynaptic role of EAT-6 at the bodywall NMJs has been well established, we found that EAT-6 may in addition regulate presynaptic ACh neurotransmission. We show that eat-6 is expressed in ventral cord ACh motor neurons, and that cell-specific RNAi of eat-6 in the ACh neurons leads to hypersensitivity to aldicarb. Electron microscopy showed an increased number of synaptic vesicles in the ACh neurons in the eat-6(ad467) mutant. Genetic analyses suggest that EAT-6 interacts with EGL-30 Gαq, EGL-8 phospholipase C and SLO-1 BK channel signaling to modulate ACh neurotransmission and that either reduced or excessive EAT-6 function may lead to increased ACh neurotransmission. Study of the interaction between eat-6 and 5-HT receptors revealed both stimulatory and inhibitory 5-HT inputs to the NMJs. We show that the inhibitory and stimulatory 5-HT signals arise from distinct 5-HT neurons. The role of eat-6 in modulation of excitatory neurotransmission by 5-HT may provide a genetic explanation for the therapeutic effects of the drugs targeting 5-HT receptors in the treatment of migraine patients.     

[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.     

Membrane instability induced by purified myelin components. Its possible relevance to experimental allergic encephalomyelitis

The fusogenic properties of purified myelin components in a system employing chicken erythrocytes were studied. Sulphatides, myelin basic protein and the apoprotein of Folch-Lees proteolipid were capable of individually inducing membrane fusion in the presence of Ca²⁺. By contrast, cerebrosides or a mixture of sulphatides and myelin basic protein (molar ratio 19 : 1) did not show such effect. The fusogenic ability of sulphatide was correlated to its behaviour in mixed monolayers with phospholipids at the air-water interface. Mixed films of sulphatides with phosphatidylcholine or sphingomyelin but not with phosphatidylethanolamine showed reductions of molecular packing and surface potential similar to those found for other fusogenic compounds. The effects of myelin components described could be of importance in the membrane instability and vesicular disruption of myelin occurring in demyelinative disorders.     

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.     

The alteration of serotonin binding sites in aged human brain

The $\text{in}$$\text{vitro}$ binding of [³H]serotonin ([³H]5-HT) to cerebral cortex from young and old adult humans was studied. With cortex from human males 23–29 years old, the binding of [³H]5-HT was a saturable process, and bound [³H]5-HT could be displaced by unlabeled 5-HT or by lysergic acid diethylamide (LSD). Two separate binding sites were discernible by Scatchard analysis. The dissociation constants were 7 nM (Kd₁) and 52 nM (Kd₂), and the total number of binding sites were 0.65 (n₁) and 2.06 (n₂) pmoles/mg protein, respectively. In cerebral cortex from aged humans (61–70 years old), the dissociation constant for [³H]5-HT binding was 198 nM, and the total number of binding sites were 4.78 pmoles/mg protein. The alteration of serotonin binding sites may be related to cerebral malfunction in old people, particularly to mental depression and sleep disturbances that commonly occur.     

Modulation of neostriatal acetylcholine in the rat by dopamine and 5-hydroxytryptamine afferents.

Endogenous and deuterium labelled acetylcholine (ACh) and choline (Ch) in the neostriatum were chemically assayed after radio-frequency lesions in the substantia nigra-ventral tegmental area and in the B7 or B8 5-hydroxytryptamine (5-HT) regions of the brainstem. Lesions in the substantia nigra-ventral tegmental region or in the B7 area, which provide dopamine (DA) and 5-HT afferents to the caudate-putamen nucleus, respectively, caused a decrease in ²H₉-ACh synthesis, while endogenous levels of ACh and Ch were unchanged. A unilateral lesion of the B8 5-HT region, which projects in part to the substantia nigra, produced an increase in endogenous ACh levels, as well as a decrease in ²H₉-ACh synthesis on the side ipsilateral to the lesion.On the basis of these data, we conclude that DA and 5-HT projections to the caudate-putamen nucleus have a net excitatory effect on cholinergic interneurons in that area. Furthermore, we suggest that the putative B8 5-HT projection is excitatory upon nigral neurons that, in turn, project to the neostriatum.     

Two Affinity States for [3H]Imipramine Binding to the Human Platelet 5-Hydroxytryptamine Carrier: An Explanation for the Allosteric Interaction Between 5-Hydroxytryptamine and Imipramine

5-Hydroxytryptamine (5-HT) showed a biphasic effect on the dissociation rate of [3H]imipramine from human platelet membranes: At low concentrations (EC50, approximately 2.5 microM), 5-HT stimulated the rate, as expected for mutually exclusive binding of 5-HT and imipramine; at higher concentrations (EC50, approximately 40 microM), 5-HT reduced this stimulated rate, a result consistent with 5-HT binding at a site that is physically distinct from both the imipramine binding site and the 5-HT transport recognition site of the 5-HT carrier. This modulatory effect could be mimicked by tryptamine, was saturable and independent of Na+ concentration, and could also be demonstrated for detergent-solubilized carriers. Monophasic association kinetics for [3H]imipramine binding were found. Heat stability experiments showed biphasic thermal inactivation curves. These results are consistent with [3H]imipramine binding to two classes of binding sites at the 5-HT carrier on human platelet membranes, with affinities three- to fivefold different. 5-HT can convert the lower-affinity state into the higher-affinity state.     

ALTERATION OF MYELIN BIOSYNTHESIS IN SLICES OF RABBIT SPINAL CORD BY ANTISERUM TO MYELIN BASIC PROTEIN AND BY PUROMYCIN 1

Abstract— Slices of rabbit spinal cord were incubated with [³H]tyrosine and [³⁵SO₄] in the presence of either 5% antiserum to myelin basic protein or 0.21 mM-puromycin. The degree of incorporation of the precursors into the basic protein (BP), the proteolipid protein (PLP) and into sulphatides, as a representative lipid, in isolated myelin was investigated. Anti-BP serum inhibited the incorporation of [³H]tyrosine into BP and PLP from 22 to 46% as compared to controls, whereas puromycin nearly completely inhibited incorporation. The incorporation of [³⁵SO₄] into sulphatides was inhibited by anti-BP serum from 20 to 34% and by puromycin from 33 to 65% as compared to controls. These alterations were myelin-specific as shown by the equal or even increased incorporation of the precursors into the homogenates of spinal cord. The results are discussed in relation to the interaction of lipids and proteins in membrane assembly.     

Pharmacological characterization of 3H-LSD binding sites in mouse brain in vivo.

In mice receiving i.v. low doses of ³H-LSD the accumulation of radioactivity in brain appears to reflect a selective binding to high affinity sites as indicated by the heterogenous regional distribution (paralleling that observed in in vitro binding studies) and by the saturable character of the process (ED₅₀ around 30μg.kg^?1 in cerebral cortex).The identity of the binding sites was assessed in various regions by administration of agonists or antagonists of different neurotransmitters. In cortex specific accumulation of ³H-LSD was easily prevented by administration of serotonin antagonists (cyproheptadine, methysergide, methiothepin) or by tryptamine derivatives (psilocin, psilocybin, dimethyltryptamine) and 5-hydroxytryptophan + pargyline. Among neuroleptics some prevented ³H-LSD binding (spiperone, haloperidol) whereas 1mg.kg^?1 pimozide was ineffective. In addition a large variety of agents (adrenergic, cholinergic, morphine) were ineffective. These data suggest a selective binding to cortical serotonin receptors.     

THE BINDING OF 5-HYDROXYTRYPTAMINE TO BUTANOL EXTRACTS OF RAT BRAIN STEM

Abstract— When butanol-water extracts of rat brain stem were incubated with [³H]5-HT, (5 × 10⁻⁷ m ), and the components resolved by chromatography on LH₂₀ Sephadex, a peak representing approximately 70% of the eluted radioactivity was found in chloroform-methanol 4:1. The peak was not found in identically prepared extracts from rat diaphragm, neither was a similar peak found when brain extracts were incubated with [¹⁴C]ACh (10⁻⁶ m ), suggesting a degree of selectivity. Binding was not saturated at concentrations of 5 × 10⁻⁵ m -5-HT. The binding was highly sensitive to the presence of water, requiring about 15% (v/v) for optimum binding. The implications of these findings are discussed in terms of a possible '5-HT receptor'.     

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons : II. Developmental aspects

Dissociated sympathetic neurons from the neonatal rat, grown in cell culture in the virtual absence of other cell types, can develop many of the properties expected of differentiated adrenergic neurons including the ability to synthesize and accumulate catecholamines (CA)². However, in the presence of high concentrations of appropriately conditioned medium (CM), the cultures develop the ability to synthesize and accumulate acetylcholine (ACh); correspondingly, their ability to synthesize CA decreases. In this paper several developmental aspects of the CM effect are described. The time course of development of cultures grown with or without CM was followed using synthesis and accumulation of [³H]CA from [³H]tyrosine and production of [³H]ACh from [³H]choline as assays for adrenergic and cholinergic differentiation. The ability to produce CA or ACh developed along parallel time courses in the two sets of cultures, rising primarily during the second week in vitro and reaching a plateau during the fourth week. When CM was used as a cholinergic developmental signal, the sympathetic neurons showed a decreasing response to addition of CM as they matured adrenergically; addition of CM during the third or fourth 10 days in vitro was not as effective in inducing ACh production as addition during the first or second 10 days. Similarly, removal of CM at various times from cultures previously grown in CM showed that the cholinergic induction caused by CM was not easily reversible in older cultures. Thus, as with the adrenergic decision, the cholinergic decision becomes less reversible as the phenotype becomes fully expressed.