HPLC analysis of putative amino acid neurotransmitters released from primary cerebellar cultures

Rogers, Keith L.; Philibert, Robert A.; Allen, Albert J.; Molitor, Jerry A.; Wilson, Elizabeth J.; Dutton, Gary R.

doi:10.1016/0165-0270(87)90012-4

Cited by 33 publications

(14 citation statements)

References 24 publications

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“…The HPLC method employed yields stable derivatives and allows the quantification of picomole quantities of the putative amino acid neurotransmitters and neuromodulators y-aminobutyric acid, glutamate, aspartate, and adenosine in addition to taurine (Rogers et al, 1987). The results of a typical 40 mM K+-stimulation time course are given in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The basic perfusion buffer consisted of 150.5 mM NaCl, 2.0 mM KC1, 2.5 mM CaCl,, 1.0 mM Mg.S04, 4.2 mM NaHC03, 0.05 mM glutamine, and 12.5 mM HEPES (pH 7.4). Here glutamine was used as an energy source (Yu and Hertz, 1983) in place of glucose, which was found to cause interference peaks in the HPLC chromatogram (Rogers et al, 1987). The chamber was connected to a peristaltic pump, and a flow rate of 0.4 ml/min was established.…”

Section: Preparation and Perfusion Of Cell Culturesmentioning

confidence: 99%

“…i o I Chromatography Endogenous taurine content was measured via HPLC (Rogers et al, 1987). Briefly, 1 day before analysis, samples were allowed to equilibrate at room temperature, redried by adding 50 pl of a solution consisting of 40% water, 40% ethanol, and 20% triethylamine (TEA), and placed under high vacuum for 2 h. Subsequently, the samples were derivatized by adding 20 pl of solution consisting of 70% ethanol, 10% water, 10% TEA, and 10% phenylisothiocyanate, vortexed, reacted at room temperature for 20 min, and placed under high vacuum for at least 6 h. On the day of analysis, samples were dissolved in 100 p1 of eluent and 70 pi injected into the HPLC.…”

Section: Preparation and Perfusion Of Cell Culturesmentioning

confidence: 99%

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Dose‐Dependent, K⁺‐Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca²⁺‐Dependent

Philibert¹,

Rogers

Allen³

et al. 1988

Journal of Neurochemistry

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The K+-stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7-9-day-old rats was studied at 16-18 days in vitro using HPLC analysis. Taurine efflux was dose-dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K+-stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3-5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0-5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.

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Section: Resultsmentioning

confidence: 99%

Section: Preparation and Perfusion Of Cell Culturesmentioning

confidence: 99%

Section: Preparation and Perfusion Of Cell Culturesmentioning

confidence: 99%

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Dose‐Dependent, K⁺‐Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca²⁺‐Dependent

Philibert¹,

Rogers

Allen³

et al. 1988

Journal of Neurochemistry

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“…Using well-defined primary cultures of neurons from the rat cerebellum and high-performance liquid chromatography (HPLC) (Rogers et al, 1987) we have looked at the evoked release of a range of endogenous amino acids in the presence of the cyclooxygenase inhibitor indomethacin and various prostanoids (PGE2, Faalpha, iloprost, and U46619). Indomethacin itself did not effect release of amino acids, nor did it affect K+-stimulated release.…”

Section: Roles For Eicosanoids In the Cnsmentioning

confidence: 99%

Astrocytes as eicosanoid‐producing cells

Murphy

Pearce

et al. 1988

Glia

123

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A variety of prostaglandins and leukotrienes, together with thromboxane and prostacyclin metabolites, can be detected in central nervous tissues and in cerebrospinal fluid. Defined cultures of astrocytes have revealed these cells to be a major source of eicosanoids. In common with other eicosanoid-producing cells, agents such as calcium ionophores and phorbol esters are potent stimuli for promoting release. While in other tissues agonists for receptors linked to calcium mobilisation prompt eicosanoid release, this does not seem to be the case in astrocytes, though a range of such receptors are present. The notable exceptions to this observation are adenosine triphosphate and adenosine diphosphate, presumably acting through P2 purinergic receptors. Many cell types in the CNS are targets for eicosanoids, possessing receptors linked to adenylate cyclase or phospholipase C. An appreciation of the functional significance of activation of these receptors is just now beginning. Eicosanoids have effects in the CNS that involve not only the vascular supply but also synaptic modulation and immune regulation.

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“…A range of precolumn HPLC [12][13][14][15][16] and CZE [17][18][19][20][21][22] based assays have been reported for the measurement of amino acids in brain tissue and microdialysis samples. The majority of these are developed for the measurement of the neuroactive amino acids.…”

Section: Introductionmentioning

confidence: 99%

Rapid high-throughput assay for the measurement of amino acids from microdialysates and brain tissue using monolithic C18-bonded reversed-phase columns

Dawson

Organ

Winter

et al. 2004

Journal of Chromatography B

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HPLC analysis of putative amino acid neurotransmitters released from primary cerebellar cultures

Cited by 33 publications

References 24 publications

Dose‐Dependent, K⁺‐Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca²⁺‐Dependent

Dose‐Dependent, K⁺‐Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca²⁺‐Dependent

Astrocytes as eicosanoid‐producing cells

Rapid high-throughput assay for the measurement of amino acids from microdialysates and brain tissue using monolithic C18-bonded reversed-phase columns

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HPLC analysis of putative amino acid neurotransmitters released from primary cerebellar cultures

Cited by 33 publications

References 24 publications

Dose‐Dependent, K+‐Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca2+‐Dependent

Dose‐Dependent, K+‐Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca2+‐Dependent

Astrocytes as eicosanoid‐producing cells

Rapid high-throughput assay for the measurement of amino acids from microdialysates and brain tissue using monolithic C18-bonded reversed-phase columns

Contact Info

Product

Resources

About

Dose‐Dependent, K⁺‐Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca²⁺‐Dependent

Dose‐Dependent, K⁺‐Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca²⁺‐Dependent