Effect of L-type and P-type Ca⁺⁺ channel blockers on the LPA-induced influx of Ca⁺⁺ into human RBCs.

(A) Conditions are the same as those described in Figure 5, except that the RBCs were pretreated for 10 minutes with (top to bottom tracings) buffer only, 50 μmol/L verapamil, 100 μmol/L nifedipine, or 5 mmol/L EGTA before stimulation with 5 μmol/L LPA. The bottom tracing displays the response of unstimulated red cells. (B) RBCs were prepared as in Figure 6A, but the pretreatment involved a 20-minute exposure to buffer only before induction with 5 μmol/L LPA (dotted line), a 20-minute exposure to 1 μmol/L ω-agatoxin before stimulation with 5 μmol/L LPA (thick line), or treatment with buffer only (thin line). After the desired LPA stimulation (approximately 10 minutes), the cells were analyzed by flow cytometry as in Figure 4. Flow cytometry was used instead of fluorescence spectroscopy to reduce the amount of ω-agatoxin required to conduct the study. Agatoxin pretreatment reduced the fraction of fluorescent cells (Ca⁺⁺ positive) from 21% to 4% in this sample. The unstimulated control RBCs contained 3% fluorescent cells.