Microanalysis of Amino Acids and Their Determination in Biological Material Using Dansyl Chloride

Briel, G.; Neuhoff, Volker; Maier, Marianne

doi:10.1515/bchm2.1972.353.1.540

Cited by 110 publications

(11 citation statements)

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“…Each preparation was stimulated once only and then discarded. The amount of aspartate, glutamate, GABA, glycine, taurine, glutamine and alanine in the samples was estimated using a double-label modification of the microdansylation procedure of Briel & Neuhoff (1972). For full details of the assay procedure, see Clark & Collins (1976).…”

Section: Methodsmentioning

confidence: 99%

Evidence of a neurotransmitter role for aspartate and γ‐aminobutyric acid in the rat olfactory cortex

Collins

1979

The Journal of Physiology

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SUMMARY1. Using a cortical cup technique, the release of seven endogenous amino acids from the isolated rat olfactory cortex slice has been monitored.2. Electrical stimulation of the lateral olfactory tract at a frequency of 4 min-was accompanied by a significant increase in the release of aspartate, GABA and taurine; the release of GABA and aspartate but not that of taurine was Ca2+-dependent.3. Chronic unilateral bulbectomy was accompanied by a specific, significant fall in the aspartate content of the olfactory cortex which reached a maximum 5 days after surgery and persisted for at least a further 5 days. Electrical stimulation of the lateral olfactory tract of such preparations did not release any of the amino acids under investigation.4. When slices from the unoperated side were exposed to solutions containing 25 mM-K+ or stimulated with electrodes placed directly on the cortex enclosed by the cup there was a Ca2+-dependent release of aspartate and GABA accompanied by a Ca2+-independent release of taurine. Following chronic bulbectomy, these procedures failed to evoke significant release of aspartate whereas the characteristics of GABA and taurine release were unaltered.5. It is concluded that aspartate may be the excitatory transmitter of some of the terminals of the lateral olfactory tract fibres and that GABA may be a transmitter at some inhibitory synapses of the rat olfactory cortex.

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Section: Methodsmentioning

confidence: 99%

Evidence of a neurotransmitter role for aspartate and γ‐aminobutyric acid in the rat olfactory cortex

Collins

1979

The Journal of Physiology

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“…Uptake is linear with time to 30 min, so provides an approximation to the initial influx velocity. (Briel & Neuhoff, 1972). The radioactive dansyl-amino acids were separated from each other and from contaminants by thin layer chromatography (Joseph & Halliday, 1975) Young et al (1973).…”

Section: Methodsmentioning

confidence: 99%

Indirect Effects of Amino‐acids on Sympathetic Ganglion Cells Mediated Through the Release of Γ‐aminobutyric Acid From Glial Cells

Bowery

Brown

Collins

et al. 1976

British J Pharmacology

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All experiments were performed on rat isolated desheathed superior cervical ganglia maintained in Krebs solution containing amino‐oxyacetic acid (10 μm) at 25°C. Influx rates of γ‐amino‐n‐butyric acid (GABA) were measured by incubating ganglia in 0.5 μm [3H]‐GABA for 30 minutes. Influx was inhibited by 50% on adding 14.3 μm unlabelled GABA, 59.2 μm β‐alanine (BALA) or 424 μm β‐amino‐n‐butyric acid (BABA). Efflux of [3H]‐GABA into non‐radioactive solution superfused over ganglia previously incubated for 60 min in 1 μm [3H]‐GABA was measured. The mean resting efflux rate coefficient (k) was 0.64 ±0.05 × 10−3 min−1. Addition of high concentrations of unlabelled GABA, BABA or BALA to the superfusing solution increased k by (maximally) 3.6–4.3 times; half‐maximal increases occurred at the following concentrations: GABA, 16 μm; BALA, 85 μm; BABA, 606 μm. Replacement of external Na+ with Li+ or TRIS increased the resting value of k and inhibited acceleration by external amino acids. Prior incubation in 1 μm [3H]‐GABA with 1 mm unlabelled GABA increased resting k 1.5 times;, but did not alter the peak rate coefficient produced by external amino acids. Neuronal depolarization produced by the amino acids was measured with surface electrodes. Preincubation in 1 mm GABA for 60 min potentiated low‐amplitude responses to BALA or BABA but not those to GABA or 3‐aminopropanesulphonic acid (a potent agonist with low affinity for the GABA carrier). Omission of external Na+ reduced responses to BABA but increased those to GABA. Incubation in 1 mm GABA for 60 min (as required to potentiate BABA or BALA actions) increased the amount of GABA in the tissue from 0.21 to 0.73 mmol/kg wet weight. Autoradiographs in which labelled GABA was used indicated that uptake into neuroglial cells was responsible for this accumulation. It is suggested that: (i) BALA and BABA are substrates for the inward GABA carrier responsible for GABA entry into ganglionic glial cells; (ii) they accelerate efflux by inhibiting carrier‐mediated re‐accumulation of effluent GABA by the glial cells; (iii) interstitial GABA concentrations are thereby increased to a level capable of depolarizing adjacent neurones; and (iv) this, rather than direct GABA‐receptor activation, accounts for the depolarization produced by low concentrations of BALA and BABA. Potentiation of their depolarizing action after pre‐incubation in 1 mm GABA is suggested to result from the increased amount of intracellular GABA available for release, and is quantitatively compatible with this increase; inhibition in Na+‐free solution is due to their inability to inhibit re‐accumulation of GABA under these conditions. A model for the action of carrier substrates is described in an Appendix. Calculations based thereon yield increments in interstitial GABA concentration in the presence of carrier substrates compatible with those determined experimentally (up to 1 μm at rest or 3.4 μm after pre‐incubation in GABA).

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“…The amino acids taurine, GABA, glycine, aspartate, glutamate, glutamine and alanine present in the Ringer solutionswere estimatedusing a modification of the highly sensitive microdansylation originally described by Briel & Neuhoff (1972). Briefly, known amounts (87,000 d.p.m.)…”

Section: Metuodsmentioning

confidence: 99%

“…per sample at a final concentration of 2-5 mM). The reaction was allowed to proceed for 30 min at 400 C in the dark (Briel & Neuhoff, 1972) and the solutions then evaporated to dryness by blowing air over the surface. The dansyl-amino acids were extracted from the salts by the addition of 6 #1. of a mixture of acetone/glacial acetic acid (3: 2, v: v) and approximately 1 #1. of the extract applied to one corner of a 375 x 375 mm polyamide thin layer chromatography (TLC) plate.…”

Section: Metuodsmentioning

confidence: 99%

“…Firstly, it has to be assumed that the labelled amino acids mix with the endogenous stores present within the brain and secondly, it is not possible to monitor the release of more than two compounds (having different radioactive labels) so the specificity of any observed release is always in doubt. These problems were partially overcome by the advent of sensitive automated amino acid analysers but it was not until the development of the highly sensitive microdansylation procedure (Briel & Neuhoff, 1972) and its subsequent modification (for example, Snodgrass & Iversen, 1973) to allow for more precise estimates that the rapid, simultaneous estimation of a large number of amino acids in picomole amounts could be undertaken. The present paper describes some experiments in which the release of endogenous taurine, GABA, glycine, aspartame, glutamate, glutamine and alanine from the visual cortex of anaesthetized rats has been monitored.…”

Section: Introductionmentioning

confidence: 99%

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The release of endogenous amino acids from the rat visual cortex.

Clark¹,

Collins²

1976

The Journal of Physiology

109

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SUMMARY1. The release of endogenous taurine, GABA, glycine, aspartate, glutamate, glutamine and alanine from the rat visual cortex was measured using a cortical cup technique. The electrocorticogram (ECoG) was monitored throughout most experiments.2. Spreading depression, evoked by the dropwise placement of 10 % KCl solution on to the brain outside the cup was associated with a significant increase in the release of GABA and glutamine but a marked fall in that of glutamate. The evoked release of GABA and glutamate but not of glutamine was Ca2+-dependent.3. A solution containing 50 mM-K+ placed within the cup elicited a significant increase in the release of taurine and GABA, whereas 100 mM-K+ additionally released aspartate and glutamate. The K+-evoked release of these amino acids with the exceptions of taurine and glutamine was Ca2+-dependent.4. Three series of experiments were carried out in which the preparations were stimulated electrically. Bipolar stimulation (100 Hz, 1 msec pulse width, 2-5 mA for 5 min) with the electrode within the cup was followed by significant increases in taurine, GABA and glutamate release; using a 5 mA current, there was an additional release of aspartate and alanine. Only the evoked release of GABA and glutamate was Ca2+-dependent.5. In the second and third series of experiments, the electrode was sited adjacent to the cup or on the contralateral cortex respectively. Following stimulation (100 Hz, 1 msec pulse width, 2-5 mA for 5 min) there was a significant increase in taurine and GABA release and a significant fall in the release of aspartate and glutamate. With the exception of taurine, these changes in release were Ca2+-dependent. Reducing the stimulus current to 1P5 mA or the period of stimulation to 2-5 min initiated similar but statistically insignificant changes in release. A range (10-100 Hz) of stimulation frequencies was examined: the evoked release of GABA was R. M. CLARK AND G. G. S. COLLINS linearly related to frequency whereas that of taurine was frequencyindependent. The fall in aspartate and glutamate release was maximal at a frequency of about 50 Hz.6. The results are discussed in relation to (a) the possible sites of release of the amino acids and (b) the proposed neurotransmitter roles of the physiologically active amino acids.

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Microanalysis of Amino Acids and Their Determination in Biological Material Using Dansyl Chloride

Cited by 110 publications

References 0 publications

Evidence of a neurotransmitter role for aspartate and γ‐aminobutyric acid in the rat olfactory cortex

Evidence of a neurotransmitter role for aspartate and γ‐aminobutyric acid in the rat olfactory cortex

Indirect Effects of Amino‐acids on Sympathetic Ganglion Cells Mediated Through the Release of Γ‐aminobutyric Acid From Glial Cells

The release of endogenous amino acids from the rat visual cortex.

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