Intracellular concentrations of Fura-2 and Fura-2/AM in vascular smooth muscle cells following perfusion loading of Fura-2/AM in arterial segments
| Autor: | C. Capdeville-Atkinson, L. Bellucci, A. Nicolas, A. Robert, Pierre Leroy, Nguyen Tran, Jeffrey Atkinson |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Male
endocrine system Vascular smooth muscle Fura-2 Physiology chemistry.chemical_element In Vitro Techniques Alkaline hydrolysis (body disposal) Calcium Muscle Smooth Vascular chemistry.chemical_compound Animals Rats Wistar Molecular Biology Chromatography High Pressure Liquid Fluorescent Dyes Detection limit Chromatography Arteries Cell Biology Fluorescence Rats Perfusion Spectrometry Fluorescence chemistry Biochemistry Intracellular |
| Zdroj: | Cell Calcium. 18:420-428 |
| ISSN: | 0143-4160 |
| DOI: | 10.1016/0143-4160(95)90057-8 |
| Popis: | A new method for the determination of tissue concentrations of Fura-2 and Fura-2/AM was developed based upon acetonitrile extraction followed by RP-HPLC separation (using tetrahexylammonium as counter-ion), post-column alkaline hydrolysis of Fura-2/AM, and fluorimetric detection. The detection limit was 1.2 nM and 1 nM for Fura-2 and Fura-2/AM, respectively. When this technique was applied to perfusion-loaded segments of the rat tail artery, intracellular concentrations of Fura-2 determined by tissue disruption were 10 times those obtained by comparing the increase in fluorescence at the isoemissive point (following loading), with a calibration curve for Fura-2. Loading conditions of 90 min at [Fura-2/AM]e = 5 microM were optimal in terms of [Fura-2]i which attained a concentration not significantly different from [Fura-2/AM]e. Under such conditions, however, Fura-2/AM also accumulated in the arterial wall. Although incompletely de-esterified, Fura-2/AM metabolites produced by in vitro incubation of Fura-2/AM with pig liver esterases could be easily detected, fluorescent forms of Fura-2 with a different sensitivity for calcium were not detected in arterial extracts. |
| Databáze: | OpenAIRE |
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