benzofurans and 6-carboxyfluorescein

The mechanism of collagen-induced human platelet activation was examined using Ca2+, Na+, and the pH-sensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Administration of a moderate dose of collagen (10 microg/ml) to human platelets resulted in an increase in [Ca2+](i) and platelet aggregation. The majority of this increase in [Ca2+](i) resulted from the influx of calcium from the extracellular milieu via the Na+/Ca2+ exchanger (NCX) functioning in the reverse mode and was reduced in a dose-dependent manner by the NCX inhibitors 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (KD(50) = 4.7 +/- 1.1 microm) and KB-R7943 (KD(50) = 35.1 +/- 4.8 microm). Collagen-induced platelet aggregation was dependent on an increase in [Ca2+](i) and could be inhibited by chelation of intra- and extracellular calcium through the administration of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) and EGTA, respectively, or via the administration of BAPTA-AM to platelets suspended in no-Na+/HEPES buffer. Collagen induced an increase in [Ca2+](i) (23.2 +/- 7.6 mm) via the actions of thromboxane A(2) and, to a lesser extent, of the Na+/H+ exchanger. This study demonstrates that the collagen-induced increase in [Ca2+](i) is dependent on the concentration of Na+ in the extracellular milieu, indicating that the collagen-induced increase in [Ca2+](i) causes the reversal of the NCX, ultimately resulting in an increase in [Ca2+](i) and platelet aggregation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aspirin; Benzofurans; Blood Platelets; Calcium; Collagen; Dose-Response Relationship, Drug; Egtazic Acid; Ethers, Cyclic; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Imidazoles; Inositol 1,4,5-Trisphosphate; Models, Biological; Organic Chemicals; Platelet Activation; Platelet Aggregation; Sodium; Sodium-Calcium Exchanger; Specimen Handling; Temperature; Thromboxane A2; Time Factors; Vasoconstrictor Agents

The new, highly fluorescent, calcium-sensitive dye, fura-2, can be loaded nondisruptively into intact cells by means of its permeant ester and used to measure the free calcium ion concentration in individual cells. For fura-2 to signal cytosolic calcium, it must be distributed homogeneously and exclusively throughout the cytoplasmic space. However, microscopic examination of bovine aortic endothelial cells loaded with fura-2 by exposure to its permeant ester reveals fluorescence associated with discrete intracellular structures rather than the homogeneous distribution expected for a cytosolic stain. Simultaneous labeling of bovine aortic endothelial cells with fura-2 and rhodamine 123 (a mitochondrial fluorescent vital stain) identifies these structures as mitochondria. Subcellular dye localizations are not observed when the cells are loaded with other putative cytosolic stains that gain access to the cytosol by means of a membrane permeant ester. Both carboxyfluorescein and indo-1 (another member of the family of second generation calcium indicators) stain the cytoplasm diffusely. It is suggested that fura-2 fluorescence accumulates in certain cells in association with mitochondria. It is important to assess the intracellular distribution of fura-2 when this indicator is used to measure the free cytosolic calcium ion concentration.

Topics: Animals; Aorta; Benzofurans; Calcium; Cattle; Cytoplasm; Endothelium; Fluoresceins; Fluorescent Dyes; Fura-2; Microscopy, Fluorescence; Mitochondria; Rhodamine 123; Rhodamines