Chemically enhanced liquid chromatography/tandem mass spectrometry determination of glutamic acid in the diffusion medium of retinal cells

Timperio, Anna Maria; Fagioni, Marco; Grandinetti, Felice; Zolla, Lello

doi:10.1002/bmc.856

Cited by 30 publications

(29 citation statements)

References 32 publications

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“…The rate of glutamate release was measured using HPLC–electrospray Tandem–Mass Spectrometry (ESI–MS/MS) in each sample, according to previous studies (Catalani et al. 2007; Timperio et al. 2007; Cervia et al.…”

Section: Methodsmentioning

confidence: 99%

Functional effects of somatostatin receptor 1 activation on synaptic transmission in the mouse hippocampus

Cammalleri

Martini

Timperio

et al. 2009

Journal of Neurochemistry

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Somatostatin-14 (SRIF) co-localizes with GABA in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of hippocampal activity has been proposed, although the exact contribution of each SRIF receptor (sst 1 -sst 5 ) in mediating SRIF action requires some clarification. We used hippocampal slices of wild-type and sst 1 knockout (KO) mice and selective pharmacological tools to provide conclusive evidence for a role of sst 1 in mediating SRIF inhibition of synaptic transmission. With single-and double-label immunohistochemistry, we determined the distribution of sst 1 in hippocampal slices and we quantified sst 1 colocalization with SRIF. With electrophysiology, we found that sst 1 activation with CH-275 inhibited both the NMDA-and the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated responses. Results from sst 1 KO slices confirmed the specificity of CH-275 effects; sst 1 activation did not affect the inhibitory transmission which was in contrast increased by sst 4 activation with L-803,087 in both wild-type and sst 1 KO slices. The AMPA-mediated responses were increased by L-803,087. Functional interaction between sst 1 and sst 4 is suggested by the finding that their combined activation prevented the CH-275-induced inhibition of AMPA transmission. The involvement of pre-synaptic mechanisms in mediating inhibitory effects of sst 1 on excitatory transmission was demonstrated by the finding that CH-275 (i) increased the paired-pulse facilitation ratio, (ii) did not influence the AMPA depolarization in the presence of tetrodotoxin, and (iii) inhibited glutamate release induced by epileptiform treatment. We conclude that SRIF control of excitatory transmission through an action at sst 1 may represent an important contribution to the regulation of hippocampal activity.

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Section: Methodsmentioning

confidence: 99%

Functional effects of somatostatin receptor 1 activation on synaptic transmission in the mouse hippocampus

Cammalleri

Martini

Timperio

et al. 2009

Journal of Neurochemistry

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“…The rate of glutamate release was measured using HPLC electrospray tandem mass spectrometry (ESI‐MS), according to previous studies (Catalani et al. 2007; Timperio et al. 2007).…”

Section: Methodsmentioning

confidence: 99%

Modulation of the neuronal response to ischaemia by somatostatin analogues in wild‐type and knock‐out mouse retinas

Cervia

Martini

Ristori

et al. 2008

Journal of Neurochemistry

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Somatostatin acts at five G protein‐coupled receptors, sst1‐sst5. In mouse ischaemic retinas, the over‐expression of sst2 (as in sst1 knock‐out mice) results in the reduction of cell death and glutamate release. In this study, we reported that, in wild‐type retinas, somatostatin, the multireceptor ligand pasireotide and the sst2 agonist octreotide decreased ischaemia‐induced cell death and that octreotide also decreased glutamate release. In contrast, cell death was increased by blocking sst2 with cyanamide. In sst2 over‐expressing ischaemic retinas, somatostatin analogues increased cell death, and octreotide also increased glutamate release. To explain this reversal of the anti‐ischaemic effect of somatostatin agonists in the presence of sst2 over‐expression, we tested sst2 desensitisation because of internalisation or altered receptor function. We observed that (i) sst2 was not internalised, (ii) among G protein‐coupled receptor kinases (GRKs) and regulators of G protein signalling (RGSs), GRK1 and RGS1 expression increased following ischaemia, (iii) both GRK1 and RGS1 were down‐regulated by octreotide in wild‐type ischaemic retinas, (iv) octreotide down‐regulated GRK1 but not RGS1 in sst2 over‐expressing ischaemic retinas. These results demonstrate that sst2 activation protects against retinal ischaemia. However, in the presence of sst2 over‐expression sst2 is functionally desensitised by agonists, possibly because of sustained RGS1 levels.

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“…Several disadvantages are associated with these pre-column derivatization methods and the analysis of their derivatives by LC-MS and LC-MS/MS: (i) long derivatization reaction time (Dansyl, 35–50 min [26], PITC, 20 min [27], FMOC, 1 hr [22], Butanol, 1 hr [22]), (ii) complex sample preparation (PITC [27]), (iii) inability to derivatize secondary amino acids (OPA [26]), (iv) derivative instability (OPA [26,28,29]; PITC [30]), (v) photosensitive adducts (Dansyl [28]), (vi) inconsistent production of derivatives (Dansyl [28]), (vii) extraction of excess reagent must be performed to stop derivatization and avoid spontaneous hydrolysis of adducts (FMOC [26,31]), (viii) removal of excess reagent is necessary to avoid rapid RPLC column deterioration (OPA [32], PITC [26,27]) and (ix) long analysis time of amino acid derivatives by LC-MS and LC-MS/MS (20–45 min [22,31,32,33,34]). These disadvantages render these derivatization methods impractical for metabolomics analysis since they introduce errors which can compromise the quality of the data.…”

Section: Introductionmentioning

confidence: 99%

An UPLC-ESI-MS/MS Assay Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization for Targeted Amino Acid Analysis: Application to Screening of Arabidopsis thaliana Mutants

2012

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In spite of the large arsenal of methodologies developed for amino acid assessment in complex matrices, their implementation in metabolomics studies involving wide-ranging mutant screening is hampered by their lack of high-throughput, sensitivity, reproducibility, and/or wide dynamic range. In response to the challenge of developing amino acid analysis methods that satisfy the criteria required for metabolomic studies, improved reverse-phase high-performance liquid chromatography-mass spectrometry (RPHPLC-MS) methods have been recently reported for large-scale screening of metabolic phenotypes. However, these methods focus on the direct analysis of underivatized amino acids and, therefore, problems associated with insufficient retention and resolution are observed due to the hydrophilic nature of amino acids. It is well known that derivatization methods render amino acids more amenable for reverse phase chromatographic analysis by introducing highly-hydrophobic tags in their carboxylic acid or amino functional group. Therefore, an analytical platform that combines the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) pre-column derivatization method with ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) is presented in this article. For numerous reasons typical amino acid derivatization methods would be inadequate for large scale metabolic projects. However, AQC derivatization is a simple, rapid and reproducible way of obtaining stable amino acid adducts amenable for UPLC-ESI-MS/MS and the applicability of the method for high-throughput metabolomic analysis in Arabidopsis thaliana is demonstrated in this study. Overall, the major advantages offered by this amino acid analysis method include high-throughput, enhanced sensitivity and selectivity; characteristics that showcase its utility for the rapid screening of the preselected plant metabolites without compromising the quality of the metabolic data. The presented method enabled thirty-eight metabolites (proteinogenic amino acids and related compounds) to be analyzed within 10 min with detection limits down to 1.02 × 10−11 M (i.e., atomole level on column), which represents an improved sensitivity of 1 to 5 orders of magnitude compared to existing methods. Our UPLC-ESI-MS/MS method is one of the seven analytical platforms used by the Arabidopsis Metabolomics Consortium. The amino acid dataset obtained by analysis of Arabidopsis T-DNA mutant stocks with our platform is captured and open to the public in the web portal PlantMetabolomics.org. The analytical platform herein described could find important applications in other studies where the rapid, high-throughput and sensitive assessment of low abundance amino acids in complex biosamples is necessary.

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Chemically enhanced liquid chromatography/tandem mass spectrometry determination of glutamic acid in the diffusion medium of retinal cells

Cited by 30 publications

References 32 publications

Functional effects of somatostatin receptor 1 activation on synaptic transmission in the mouse hippocampus

Functional effects of somatostatin receptor 1 activation on synaptic transmission in the mouse hippocampus

Modulation of the neuronal response to ischaemia by somatostatin analogues in wild‐type and knock‐out mouse retinas

An UPLC-ESI-MS/MS Assay Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization for Targeted Amino Acid Analysis: Application to Screening of Arabidopsis thaliana Mutants

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