Day-night rhythm of 5-methoxytryptamine biosynthesis in the pineal gland of the golden hamster (Mesocricetus auratus).

5-Methoxytryptamine is a potent agonist of presynaptic 5-hydroxytryptamine autoreceptors modulating serotonin release in the central nervous system. This methoxyindole can be synthesized in the pineal gland, but its presence in vivo is still controversial, probably because of rapid catabolism by monoamine oxidase. An improved high-pressure liquid chromatography method, with coulometric detection, has been developed for the simultaneous measurement of melatonin, 5-methoxytryptamine, 5-methoxytryptophol and 5-methoxyindolacetic acid. We have demonstrated a day-night rhythmicity in the amount of 5-methoxytryptamine in the pineal gland of golden hamsters (Mesocricetus auratus) maintained under a long photoperiod (14 h light: 10 h darkness) and pretreated with the monoamine oxidase inhibitor pargyline. Levels of 5-methoxytryptamine were highest at 16.30 h and lowest at 00.30 h. The rhythm for 5-methoxytryptamine appears to be the same as for serotonin (opposite in phase to that of melatonin). The identification of 5-methoxytryptamine has been confirmed by analysis with gas chromatography-mass spectrometry.

A common binding site for tricyclic and nontricyclic 5-hydroxytryptamine uptake inhibitors at the substrate recognition site of the neuronal sodium-dependent 5-hydroxytryptamine transporter.

An investigation of the site of interaction of a variety of tricyclic and nontricyclic 5-HT uptake inhibitors with the neuronal sodium-dependent 5-HT transporter was undertaken. The dissociation of [3H]paroxetine binding induced by indalpine (10 microM), SL 81.0385 (10 microM), fluoxetine (10 microM), citalopram (10 microM), paroxetine (0.15 microM), imipramine (10 microM) and 5-HT (50 microM) produced monophasic dissociation curves and gave t1/2 values of dissociation similar to that induced by dilution alone. In inhibition studies of [3H]paroxetine binding with citalopram, imipramine and 5-HT, increases in the concentration of [3H]radioligand used led to parallel rightward shifts of the inhibition curves with no diminution of the maximum degree of inhibition (Imax). "Schild-type" analyses of the data obtained from the inhibition curves with these 3 compounds gave slopes close to unity. In chemical modification studies, treatment of membrane fractions with N-ethylmaleimide led to a pronounded reduction in specific [3H]paroxetine binding. Preincubation of these membranes with SL 81.0385, fluoxetine, imipramine, tryptamine and 5-HT provided significant protection against this NEM-induced inactivation. The above findings are interpreted to provide evidence for a common or at least overlapping binding site for the tricyclic and nontricyclic 5-HT uptake inhibitors with the substrate recognition site of the neuronal sodium-dependent 5-HT transporter.

Pharmacological characterization of the cloned human 5-hydroxytryptamine transporter.

We performed an extensive pharmacological study of the 5-hydroxytryptamine (5-HT) transporter polypeptide cloned from human placenta. Transient expression of this 630 amino acid polypeptide in HeLa cells led to saturable 5-HT uptake activity (Km = 858 nM). This 5-HT uptake was blocked by selective 5-HT inhibitors, such as citalopram, litoxetine, sertraline, and indalpine, with Ki values in the low nanomolar range, and it exhibited a pharmacological profile similar to that found in rat brain. [3H]Citalopram binding to membrane preparations of the transfected cells occurred to a single class of high-affinity binding sites (Kd = 5.3 nM) and was potently inhibited by selective 5-HT uptake inhibitors. The pharmacological profile of [3H]citalopram binding to these transfected cells showed a good correlation with that of [3H]paroxetine binding to the rat cerebral cortical 5-HT transporter (r = 0.79). These data confirm that the full pharmacological characteristics of the 5-HT transport system are conferred by the expression of the 630 amino acid human placental 5-HT transporter polypeptide. [3H]Citalopram should, therefore, provide a useful probe for more insights at a molecular level into this cloned 5-HT transport system.

Determination of melatonin in blood plasma, using capillary gas chromatography and electron impact medium-resolution mass spectrometry.

Determination of the low concentrations (less than 10-100 pg ml-1) of melatonin in blood plasma by gas chromatography/mass spectrometry has previously required the use of negative ionization (electron capture) mass spectrometry, a technique particularly delicate to set up. We show that a general-purpose medium-resolution instrument gives adequate sensitivity in electron impact mode, when used with a capillary gas chromatographic column-switching device. A straightforward extraction procedure gives good specificity, and the limit of useful measurement is better than 10 pg per 1 ml sample.

Characterization and purification of the neuronal sodium-ion-coupled 5-hydroxytryptamine transporter.

The selective 5-hydroxytryptamine (5-HT) uptake inhibitor, [3H]paroxetine, has been used as a specific probe in a series of experiments aimed at characterizing the substrate and 5-HT uptake inhibitor binding domains on the neuronal sodium-ion-coupled 5-HT transporter. In addition, an affinity-chromatographic protocol which provided a greater than 3000-fold purification of this neuronal transporter protein is outlined.

Partial purification and characterization of the sodium-ion-coupled 5-hydroxytryptamine transporter of rat cerebral cortex.

A procedure for the extensive purification of the Na(+)-coupled 5-hydroxytryptamine transporter of rat cerebral cortex has been developed. The 5-hydroxytryptamine transporter was solubilized with the non-ionic detergent digitonin, and the detergent extracts were subjected to sequential affinity chromatography on a citalopram-based agarose support and wheat-germ-agglutinin-Sepharose. 5-Hydroxytryptamine transporters in the affinity-purified preparation were identified by using the selective 5-hydroxytryptamine-uptake inhibitor [3H]paroxetine, and were shown to display a similar pharmacological profile to those present in particulate preparations. An overall transporter purification of around 2000-fold was achieved with a 9% recovery. SDS/PAGE of affinity-chromatographed material starting from detergent extracts incubated in the presence or absence of 1 mM-citalopram indicated that a polypeptide of M(r) 73,000 corresponded to the 5-hydroxytryptamine-transporter protein.

5-methoxytryptoline and close analogs as candidates for the endogenous ligand of the 3H-imipramine recognition site.

3H-Imipramine labels with high affinity a site associated with the macromolecular complex of the serotonin transporter in brain and platelets. There is a good correlation between the potencies of drugs at inhibiting 3H-5HT uptake and at inhibiting 3H-imipramine binding. Dissociation experiments indicate that the site labelled by 3H-imipramine is not identical with the substrate recognition site of the serotonin transporter and thus, it appears that 3H-imipramine labels a modulatory site for the 5HT transport system. On this basis, the existence of an endacoid acting on the 3H-imipramine recognition site to modulate 5HT uptake is discussed. The possibility that 5-methoxytryptoline or a closely related analog may be the endogenous ligand for the 3H-imipramine recognition site is analysed on the basis of the fact that 5-methoxytryptoline is a potent inhibitor of 3H-imipramine binding that also inhibits 3H-5HT uptake.