Effect of lipid-derived second messengers on the kinetics of Ca⁺⁺ influx into human erythrocytes.

Washed human erythrocytes loaded with Fluo-3/AM and suspended in HEPES buffer (125 mmol/L NaCl, 3 mmol/L KC1, 2 mmol/L CaCl₂, 1 mmol/L MgCl₂, 1.2 mmol/L sodium phosphate, 10 mmol/L glucose, 16 mmol/L HEPES, pH 7.4) were stirred at 0.07% hematocrit in a fluorescence spectrophotometer and treated at time (t = 60 seconds) with 5 μmol/L LPA (top tracing), 14 μmol/L PA (middle tracing), 10 μmol/L Sph-1-P (lower tracing), or 5 mmol/L EGTA followed by 5 μmol/L LPA (lowest tracing). All lipid stimulants were added from stock solutions prepared in PBS. The increase in fluorescence (λ_ex = 506 nm, λ_em = 530 nm) caused by Ca⁺⁺ binding to Fluo-3 was then monitored as a function of time. Labeled RBCs not treated with any stimulant displayed no change in Fluo-3 fluorescence.