CYTOSOLIC CA2- EFFECT OF PROTEIN ADSORPTION( CONCENTRATION DETERMINATIONS IN NEUTROPHILS STIMULATED BY MONOSODIUM URATE AND CALCIUM PYROPHOSPHATE CRYSTALS )

Objective. To develop methods to correct for light scattering artifact s produced by monosodium urate monohydrate (MSUM) and calcium pyrophos phate dihydrate (CPPD) crystals in the measurement of the levels of cy tosolic free calcium, [Ca2+](i), in neutrophils and to quantitate the extent of association of neutrophils with crystals. Methods. The assoc iation of neutrophils with crystals through binding of crystals onto t he neutrophil plasma membrane and/or internalization was quantitated b y mixing crystals with diphenyl hexatriene (DPH) loaded neutrophils an d monitoring the fluorescence intensity of supernatants (''free'' neut rophils) following gravity sedimentation of ''free'' crystals and neut rophils with bound/internalized crystals. The effects of precoating CP PD and MSUM crystals with IgG, plasma, serum and BSA on crystal-neutro phil association and [Ca2+](i) were determined. Results. MSUM produced a large increase in [Ca2+](i) that reached maximal values in less tha n 1 min, whereas CPPD produced a smaller and slower increase in [Ca2+] (i). IgG adsorbed onto CPPD increased the rate and extent of increase in [Ca2+](i). Heparinized plasma and serum adsorbed onto CPPD increase d the extent but not the rate of increase in [Ca2+](i). Conclusion. [C a2+](i) increases induced by MSUM were not affected by IgG, serum or h eparinized plasma precoating. IgG, serum, or heparinized plasma adsorb ed into CPPD amplified neutrophil activation increasing the extent of the increase in [Ca2+]. Neither MSUM nor CPPD induced increases in [Ca 2+](i) were sensitive to precoating crystals with bovine serum albumin .