Further studies of the effects of diamide and hydrogen peroxide on calcium signaling in the human platelet

Intracellular calcium levels were measured in Fura-2/AM loaded human platelets. Diamide (30 mu M) reduced the Ca2+ response to serotonin (1 mu M), but not to 4-bromo-A23187 (1 mu M). The small reduction in the maximum fluorescence produced by diamide was not mirrored by a change in the sensitivity of Fura-2 pentapotassium salt to Ca2+. Changes in the spectral properties of Fura-2 thus cannot wholly explain the reduced serotonin response following diamide treatment. Hydrogen peroxide (500 mu M) produced a steady increase in the observed fluorescence ratio that was partially inhibited by the phospholipase C inhibitor U-73122 (1.5 mu M). In the absence of platelets, H2O2 reduced the Ca2+-sensitivity of Fura-2 pentapotassium salt, although it is not clear the extent to which this effect contributes to the reduction in the Ca2+ response to serotonin seen after H2O2. It is concluded that some caution may be warranted in the interpretation of the effects of H2O2 upon receptor-mediated Ca2+ signaling as measured using Fura-2. (C)1999 Prous Science. All rights reserved.