This study examined the effect of overloading human platelet intracellular Ca2+ stores on the rate of agonist-evoked external Ca2+ entry. To overload the Ca2+ stores (presumably dense tubules), external Na(+)-dependent Ca2+ efflux via Na(+)-Ca2+ exchange was inhibited by pretreating the cells with ouabain or Na(+)-free medium. Ca2+ regulation was then examined after exposure to thrombin, ADP and thapsigargin. Cytosolic free Ca2+ levels were monitored using the fluorescent probe fura-2 and external Ca2+ influx was assessed by the rates of extracellular Mn2+ or 45Ca2+ uptake. 2. Both ouabain and Na(+)-free pretreatments caused a slight increase in the resting cytosolic free Ca2+. 3. In 1 mM Ca(2+)-containing medium, Ca(2+)-overloaded platelets showed similar thrombin-evoked cytosolic free Ca2+ responses to those of control platelets. However, in Ca(2+)-free medium, they showed substantially greater thrombin-evoked cytosolic free Ca2+ responses than control platelets. Moreover, increased thrombin-evoked Ca2+ mobilization from Ca2+ storage sites was accompanied by a diminished rate of thrombin-evoked external Ca2+ entry. 4. Similar reductions in the rate of external Ca2+ entry were observed after treatment with ADP and thapsigargin. 5. Protein kinase C inhibitors (calphostin C and staurosporine) failed to reverse the effect of ouabain pretreatment on thrombin-induced changes in the cytosolic free Ca2+ response. 6. Inositol 1,4,5-trisphosphate profiles in ouabain-treated and non-treated platelets were not significantly different. 7. These data indicate that increased Ca2+ in the dense tubules is associated with diminished agonist-evoked external Ca2+ influx.
