The total and free concentrations of Ca2+ and Mg2+ inside platelet secretory granules. Measurements employing a novel double null point technique.
The inorganic ion contents of platelet alpha-granules were determined by a combination of neutron activation analysis and flame photometry. Total concentrations were estimated using intragranular water spaces measured by isotope dilution. To measure the free concentrations of Ca2+ and Mg2+ we developed a novel double null point titration technique. The method requires independent determinations of transmembrane delta pH and the availability of two divalent cation-H+ exchange ionophores with different Ca2+/Mg2+ selectivity ratios. A23187 and the halogenated analog 4-bromo-A23187 were used for this purpose. Absolute delta pH was measured by methylamine distribution, while relative pH changes induced by the ionophores were monitored with 9-aminoacridine. The free concentrations of Ca2+ and Mg2+ were found to be 12 and 326 microM, respectively. These values are markedly lower than the calculated total concentrations of these cations, i.e. 32 mM for Ca2+ and 172 mM for Mg2+.
D000818:Animals; D001792:Blood Platelets / Q000648:ultrastructure*; D000001:Calcimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000097:blood*; D003594:Cytoplasmic Granules / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000097:blood*; D008433:Mathematics; D008722:Methods; D013552:Swine
D000001:Calcimycin; C033170:4-bromo-A-23187; D008274:Magnesium; D002118:Calcium