Further Studies on the Endogenous Serotonin-Uptake-Inhibitor-Like Substances in the Human Cerebrospinal Fluid

Abstract

ABSTRACT-Theproperties of endogenous substances that inhibit 5HT uptake in human cerebrospinal fluid (CSF) were investigated. Human CSF was loaded onto a Sephadex G-25 column, and each fraction was tested for its ability to inhibit [3H]paroxetine binding in monkey brain preparations. We found four different inhibitory peaks with respective molecular weights (M.W.) of > 12400, 2000 and two of < 1350. The third and fourth peaks (F-3, F-4: < 1350 M.W.) of inhibitory activity were determined to consist of some monoamines (5HT, etc.) or their metabolites (5HIAA, etc.) and other unidentified compounds by us ing an HPLC-electrochemical detector. (13, 14). In this study we report more detailed investiga tions of the properties of the selective 5HT uptake inhibi tor-like substances in human CSF. MATERIALS AND METHODSMonkeys were anesthetized with ketaral (30 mg/kg, s.c.), and their brains were quickly removed after withdrawing blood. Fifty milliliters of human CSF sam ples were obtained from 25 patients in the course of rou tine diagnostic lumbar puncture and were collected in the Central Laboratory of Medicine. The monkey brains and the pooled human CSF were stored at -80°C until used. Gel filtrationFive milliliters of evaporated human CSF sample was chromatographed in a Sephadex G-25 column (1.9 x 87 cm) with 1 mM phosphate buffer, pH 7.4, at a flow rate of 103 ml/hr, and effluent fractions of 5.15 ml/tube were col lected. Column effluents were monitored by measuring ab sorbances at 280 nm. Aliquots (100µl) of each fraction were assayed for the ability to displace [3H]paroxetine binding. The molecular weights (M.W.s) of endogenous 5HT-uptake-inhibitor-like substances were estimated by chromatography in the same column calibrated with mark ers of known M.W. Paroxetine binding assay (15)Monkey frontal cortices were homogenized in 25 vol. of ice-cold 50 mM Tris-HC1 buffer (containing 100 mM NaCI and 5 mM KCI, pH 7.4). The P2 fractions obtained by centrifugation of this homogenate were used as the crude membrane preparations (final concentration of ap proximately 0.1 mg protein/tube) for the assay. Aliquots of crude membrane suspension were incubated with[3H]paroxetine at 221C at a final volume of 250,u1 for 180 min. Fluoxetine at 10,uM final concentration was used to determine non-specific binding. The incubation was termi nated by rapid filtration of the membrane suspension un der reduced pressure through Whatman GF/B glass fiber filters. Each filter was rapidly washed three times with 5 ml of ice-cold 50 mM Tris-HC1 buffer, pH 7.4. The filters were then dried and the radioactivities were determined in Triton X-100-toluene scintillation fluid in a liquid scintilla tion spectrometer. Monoamine uptake assay The study of [3H]5HT, [3H]norepinephrine (NE) or[3H]dopamine (DA) uptake into synaptosomes was con ducted according to the method of Snyder and Coyle (16), but with minor modifications. The monkey brains (bilateral frontal cortex) were homogenized in 0.32M sucrose, and the crude synaptosomes were obtained by different...

Effect of Long-Term Fluvoxamine Treatment on Endogenous Serotonin Uptake Inhibitor-Like Substance in Monkey Cerebrospinal Fluid

“…We found five different effluent fractions that inhibited [3H]-paroxetine binding. This profile was very similar to that which we obtained for human CSF (9). On the basis of it's Ve/Vo ratio, the first fraction may contain a high molecular weight protein ( Fig.…”

“…We previously reported that human CSF contains substances that modulate [3H]-paroxetine binding sites and 5-HT uptake sites and that the molecular weight of one of these substances was about 2000 (9). However, there are no studies on the changes in the amounts of these endogenous substances in depressive animal models.…”