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 microM) reduced the Ca2+ response to serotonin (1 microM), but not to 4-bromo-A23187 (1 microM). 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 microM) produced a steady increase in the observed fluorescence ratio that was partially inhibited by the phospholipase C inhibitor U-73122 (1.5 microM). In the absence of platelets, H2O2 reduced the Ca(2+)-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.
Department of Pharmacology and Clinical Neuroscience, Umeå University, Sweden.
H L
HL
Hedin
C J
CJ
Fowler
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood; D020013:Calcium Signaling / Q000187:drug effects*; D003958:Diamide / Q000494:pharmacology*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D066298:In Vitro Techniques; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D005456:Fluorescent Dyes; D013439:Sulfhydryl Reagents; D003958:Diamide; C049925:fura-2-am; D006861:Hydrogen Peroxide; D002118:Calcium; D016257:Fura-2