Nedathur Narasimhachari

Tryptophan hydroxylase: increase in activity by electrical stimulation of serotonergic neurons

Electrical stimulation of the rat midbrain dorsal raphe nucleus, which contains clusters of 5-HT containing cell bodies, increased the activity of tryptophan hydroxylase prepared in low speed supernatant extracts from cerebral cortex, a region containing 5-HT projections arising from the dorsal raphe nucleus. In contrast, activity of enzyme from hippocampus, which receives its 5-HT innervation primarily from the median raphe was unaffected by dorsal raphe stimulation. The activity of cortical enzyme increased maximally at a stimulation frequency between 10 and 15 Hz. The increase in activity took 20 min to reach maximum at a stimulation frequency of 10 Hz and had returned to control levels 30 min after cessation of stimulation. The increase in enzyme activity is associated with a significant increase in V(max) without any change in K(m) for substrate tryptophan or the artificial reduced pterin cofactor, d-6-methyl-5,6,7,8-tetrahydropterin. These findings may provide a basis for the increase in synthesis of 5-HT which occurs in response to nerve stimulation through the enhanced conversion of tryptophan to 5-hydroxytryptophan.

Activation of tryptophan hydroxylase by stimulation of central serotonergic neurons

Electrical stimulation of serotonergic neurons in the raphe nuclei of the midbrain or medulla enhances the formation of serotonin in vivo in the terminal projections of these neurons [I]. The acceleration of serotonin synthesis results from an increase in the conversion of tryptophan to 5-hydroxytryptophan (5-HTP) by tryptophan hydroxylase [2,3] [EC 1.14.16.4 tryptophan-5-monooxygenase: L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)], the initial and rate-limiting enzyme in the reaction sequence leading to the formation of serotonin. It is likely that this increase in serotonin synthesis is due to an activation of tryptophan hydroxylase itself since this has previously been shown to occur with in vitro depolarization of rat brain slices [4-7]. Earlier attempts to demonstrate an increase in tryptophan hydroxylase activity in vitro after in vivo electrical stimulation of serotonergic nuclei have, however, been unsuccessful [3]. We now present preliminary evidence that in vivo electrical stimulation of the serotonergic dorsal raphe nucleus produces an increase in the activity of cortical tryptophan hydroxylase, measured in vitro, that parallels increases both in the in vivo to 5-HTP (in the presence of an inhibitor of aromatic amino acid accumulation of 5-hydroxyindoleacetic acid (5-HIAA), the conversion of tryptophan decarboxylase) and in the metabolite of serotonin. In these experiments, male Sprague-Dawley rats (450-550 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a small animal stereotaxic apparatus (David Kopf Instruments). Stimulating electrodes (size O0 insect pins from Carolina Biological Supply), insulated to within 0.5 mm of the tip, were positioned in the dorsal raphe nucleus according to coordinates from the atlas of Pellegrino et al. [8] (anterior-posterior, +0.3 mm; lateral, 0 mm; depth, -1.3 mm). The animals were grounded by a probe in the rectum. Stimulation consisted of constant current menopolar square waves (200 uamps, 1.5 msec pulse width, 10 Hz) delivered from a Grass S-48 stimulator coupled to a Grass Constant Current Unit. Experimental animals received 30 min of continuous stimulation while shams were unstimulated. In one group of animals in vivo accumulation of 5-HTP was measured in order

Determination of 13 Catecholamines, Indoleamines, Metabolites and Precursors in Less Than 20 Minutes During a Single HPLC Run

Abstract Thirteen catecholamines and indoleamines are rapidly separated during a single HPLC run. They include epinephrine, norepinephrine, dopamine, serotonin, the major metabolites of these compounds and their immediate precursors. The technique was developed in order to analyze several biogenic amines in brain tissues simultaneously but the methodology has universal application. The separation is based on a fine balance between organic solvent, ion pairing reagent, buffers, pH, choice of column and flow rate.

Simultaneous HPLC Analysis of Catecholamines and Indoleamines in Mouse Brain Tissue Following Acetate Extraction and Treatment with Ascorbate Oxidase

Abstract A refined HPLC Method for the determination of monoamine levels in six brain regions is presented. Analyses are made for the olfactory tubercles, prefrontal cortex, septum, striatum, amygdala and hypothalamus of adult male ICR mice. This system permits the simultaneous analysis of norepinephrine, dopamine, serotonin and their major metabolites during a single run of approximately twenty-five minutes without prior clean-up of samples.

Activation of cortical tryptophan hydroxylase by acute morphine treatment: blockade by 6-hydroxydopamine

Acute morphine produced a dose-dependent, naloxone-sensitive, reversible increase in tryptophan hydroxylase activity in low speed supernatants of midbrain, pons-medulla and cerebral cortex but not spinal cord. The increase in cortical enzyme activity was blocked by 6-hydroxydopamine pretreatment, could be reversed in vitro by incubation with alkaline phosphatase and was non-additive with the increase in enzyme activity induced in the presence of phosphorylating conditions. Morphine administration produced an increase in Vmax but no change in Km of cortical enzyme for substrate, tryptophan, or the artificial reduced pterin cofactor, 6-methyl-5,6,7,8-tetrahydropterin. The failure of morphine to increase spinal tryptophan hydroxylase activity despite enhancement of enzyme activity in medulla indicates regional differences in responsiveness of the enzyme to in vivo activation.

Quantitation of Serotonin in Human Plasma, Serum and Cerebrospinal Fluid Samples by HPLC-EC Using 6-Hydroxytryptamine as an Internal Standard

Abstract A simple method for sample clean up and concentration of serotonin (5-HT) in biological samples, such as human cerebro-spinal fluid and serum, is described. To the sample 6-hydroxy-tryptamine (6-HT) is added as an internal standard and it is then absorbed either on C18 SEP-PAK cartridge or Biorex-70 short column. 5-HT and 6-HT are then eluted from the column with methanolic formic acid. After evaporation, the residue is dissolved in the mobile phase and an aliquot is used for LC-EC quantitation.

Effect of clonidine on the activity of tryptophan hydroxylase from rat brainstem following in vivo or in vitro treatment

In vivo administration of clonidine hydrochloride (Catapres) via tail vein injection produced a rapid increase in brainstem tryptophan hydroxylase activity assayed in vitro under subsaturating conditions of reduced pterin cofactor, 6MPH4. Enzyme activity returned to and remained at control levels about 60 min after treatment with low doses of clonidine (5 micrograms/kg). However, with higher doses of the drug (15 micrograms/kg), enzyme activity fell to below control levels for about an hour. Incubation of brainstem slices with clonidine also produced a dose-dependent increase in enzyme activity. The increase in enzyme activity appears to be mediated indirectly since it was abolished when brain catecholamine levels were depleted by pretreatment with 6-hydroxydopamine 8 days prior to clonidine treatment. The kinetic properties of tryptophan hydroxylase prepared 25 and 90 min after clonidine administration indicate that the initial increase and subsequent decrease in enzyme activity seen under these conditions may be due to changes in apparent Vmax of the enzyme.

An Extraction Method to Quantitate Serotonin in Human Serum, Amniotic Fluid and Urine Samples by HPLC Using 6-Hydroxy Tryptamine as Internal Standard

Abstract A method for extracting serotonin (5-HT) from human serum, amniotic fluid and urine samples is described. A mixture of sample, 6-hydroxy tryptamine (6-HT) as internal standard and sodium carbonate is extracted with ethyl acetate. The ethyl acetate layer is mixed with 200 ul of triethylamine phosphate buffer (pH 3.0) used for mobile phase and centrifuged. An aliquot of the aqueous layer was used for HPLC quantitation with amperometric detection. With this method 30 samples can be assayed in 6 hours.

A Micro Method for the Quantitation of Tryptophan in Biological Fluids by HPLC-EC

Abstract A micromethod for the quantitation of tryptophan in biological fluids, such as whole blood, RBC, serum, plasma or tissue homogenates using HPLC and electrochemical detection is described. -Methyl tryptophan is used as an internal standard. Ten microliters of the sample are added to a mixture of 50 ul of acetonitrite and 10 ul of internal standard (20 ng/ul). This mixture is then diluted with 180 ul of triethylamine phosphate-buffer (pH 3.0). After centrifugation 25 ul of the sample are injected into HPLC system. Standards containing 100 ng and 200 ng of tryptophan are similarly prepared and 25 ul aliquot injected into HPLC.

Tryptophan Determination in Tissue Homogenates and Biological Fluids Using HPLC-EC

Abstract A method for quantitating tryptophan in tissue homogenates and biological fluids using 6-hydroxy-tryptamine as internal standard is described. Tryptophan in CSF and free tryptophan in serum or plasma can be quantitated by directly injecting CSF or ultrafiltrate into the HPLC system with the internal standard. Serum, plasma, amniotic fluid, and saliva are deproteinized by addition of equal volume of acetonitrile. Urine samples are processed through Biorex-70 columns. Cross validation was done by comparing the values of column procedures with direct injection method.