Intracellular concentrations of fura-2 and fura-2/am in vascular smooth muscle cells following perfusion loading of fura-2/am in arterial segments.
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.
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Laboratoire de Pharmacologie Cardio-vasculaire, URA CNRS 597, Faculté des Sciences Pharmaceutiques et Biologiques, Université Henri Poincaré-Nancy 1, France.
Leroy
P
P
Bellucci
L
L
Robert
A
A
Nicolas
A
A
Atkinson
J
J
Capdeville-Atkinson
C
C
D000818:Animals; D001158:Arteries / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis*; D016257:Fura-2 / Q000031:analogs & derivatives / Q000032:analysis* / Q000302:isolation & purification; D066298:In Vitro Techniques; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010477:Perfusion; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; C049925:fura-2-am; D016257:Fura-2