Abstract：
: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.
journal_name
Cell Calciumjournal_title
Cell calciumauthors
Tran NN,Leroy P,Bellucci L,Robert A,Nicolas A,Atkinson J,Capdeville-Atkinson Cdoi
10.1016/0143-4160(95)90057-8subject
Has Abstractpub_date
1995-11-01 00:00:00pages
420-8issue
5eissn
0143-4160issn
1532-1991pii
0143-4160(95)90057-8journal_volume
18pub_type
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