Cytosolic Ca2+ concentration determinations in neutrophils stimulated by monosodium urate and calcium pyrophosphate crystals: effect of protein adsorption.
OBJECTIVE
To develop methods to correct for light scattering artifacts produced by monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) crystals in the measurement of the levels of cytosolic free calcium, [Ca2+]i, in neutrophils and to quantitate the extent of association of neutrophils with crystals.


METHODS
The association of neutrophils with crystals through binding of crystals onto the neutrophil plasma membrane and/or internalization was quantitated by mixing crystals with diphenyl hexatriene (DPH) loaded neutrophils and monitoring the fluorescence intensity of supernatants ("free" neutrophils) following gravity sedimentation of "free" crystals and neutrophils with bound/internalized crystals. The effects of precoating CPPD and MSUM crystals with IgG, plasma, serum and BSA on crystal-neutrophil association and [Ca2+]i were determined.


RESULTS
MSUM produced a large increase in [Ca2+]i that reached maximal values in less than 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 increased the extent but not the rate of increase in [Ca2+]i.


CONCLUSIONS
[Ca2+]i increases induced by MSUM were not affected by IgG, serum or heparinized plasma precoating. IgG, serum, or heparinized plasma adsorbed into CPPD amplified neutrophil activation increasing the extent of the increase in [Ca2+]. Neither MSUM nor CPPD induced increases in [Ca2+]i were sensitive to precoating crystals with bovine serum albumin.
Burt
H M
HM
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Jackson
J K
JK
D000327:Adsorption; D001798:Blood Proteins / Q000493:pharmacokinetics*; D002118:Calcium / Q000378:metabolism*; D002131:Calcium Pyrophosphate / Q000494:pharmacology*; D003460:Crystallization; D003600:Cytosol / Q000378:metabolism*; D005453:Fluorescence; D016257:Fura-2; D007425:Intracellular Membranes / Q000378:metabolism; D008027:Light; D009504:Neutrophils / Q000378:metabolism* / Q000502:physiology; D009994:Osmolar Concentration; D012542:Scattering, Radiation; D014527:Uric Acid / Q000494:pharmacology*
D001798:Blood Proteins; D014527:Uric Acid; D002118:Calcium; D016257:Fura-2; D002131:Calcium Pyrophosphate