![Fig. 3. Effect of eicosanoids on erythrocyte cytosolic Ca++. / RBCs loaded with fura-2 am (0.1% hematocrit in HEPES buffer) were placed in a quartz cuvette with stirring (37°C, 300 rpm) for fluorescence measurements (“Materials and methods”). Fluorescence was determined with excitation wavelengths 340 and 380 nm and emission wavelength 510 nm in a spectrofluorimeter at 1-second intervals for 10 minutes. The maximum cytosolic Ca++concentration ([Ca++]i) in the erythrocytes was examined under basal conditions and following addition of the indicated concentrations of U46 619 (TXA2 analog), free-arachidonic acid, 12-HETE, PGI2, or solvents. Ionomycin (1 μM) served as positive controls (“Materials and methods”). U46 619 and free-AA dose dependently increased [Ca++]i of erythrocytes. In contrast, addition of PGI2 (0.5-2 μM) or 12-HETE (0.1-1 μM) did not increase [Ca++]i over solvent control (not shown). Ionomycin (1 μM) produced an immediate increase in [Ca++]i of 105 ± 10 nM (n = 15). Data are mean ± SEM of 6 to 20 determinations in different donors. *P < .001 for either U46 619 or AA versus control. Significance was determined by Student t test.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/99/11/10.1182_blood.v99.11.3978/6/h81122629003.jpeg?Expires=1735508118&Signature=HAuPmJZc6S4-rfKqb9yLyHyRGlIMS3wt4LFjLWrzKnx-EN8AG3ctQmRblMGBcHfc17GxN0NErohEF4unnIvYbe5RuKArlLV94kt2zvPbkKMRriZKemZ8CwCInvMaP3CRlEPqpkqpHFf8U1LVmwpYlI9C9sX~QjMf4VeS61f2ptPykvKJkkx67T5Ft-iZYOhzz0fImg0-T77eSwKguxNCS-2gN1g7GFfgTsKMgWZx4lslucbOSOaq7hCLakH1~yJzqgRKWdxE6TG6e~bCxKSag8ZSvtuKu6vk4-mEHhMGM55VmZhW19cvGKj4ErKvdNn6QUs0kpClXN~1SWFNL9asDw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of eicosanoids on erythrocyte cytosolic Ca++.
RBCs loaded with fura-2 am (0.1% hematocrit in HEPES buffer) were placed in a quartz cuvette with stirring (37°C, 300 rpm) for fluorescence measurements (“Materials and methods”). Fluorescence was determined with excitation wavelengths 340 and 380 nm and emission wavelength 510 nm in a spectrofluorimeter at 1-second intervals for 10 minutes. The maximum cytosolic Ca++concentration ([Ca++]i) in the erythrocytes was examined under basal conditions and following addition of the indicated concentrations of U46 619 (TXA2 analog), free-arachidonic acid, 12-HETE, PGI2, or solvents. Ionomycin (1 μM) served as positive controls (“Materials and methods”). U46 619 and free-AA dose dependently increased [Ca++]i of erythrocytes. In contrast, addition of PGI2 (0.5-2 μM) or 12-HETE (0.1-1 μM) did not increase [Ca++]i over solvent control (not shown). Ionomycin (1 μM) produced an immediate increase in [Ca++]i of 105 ± 10 nM (n = 15). Data are mean ± SEM of 6 to 20 determinations in different donors. *P < .001 for either U46 619 or AA versus control. Significance was determined by Student t test.
