Ratiometric fluorescence calcium imaging of mouse dermal endothelial cells. Fura-2–loaded cells were stimulated with the agonists indicated. Images (original magnification, × 200) at peak increase in [Ca]_i are shown. Pseudocolor scales used to indicate the fura-2 340:380 fluorescence ratio are at right. Ratios greater than 20 are shown as white. (A) Thrombin (10 nM) triggered responses in more than 90% of wild-type endothelial cells and Par4–/– endothelial cells. Smaller responses were noted in approximately 30% of Par1–/– endothelial cells. Thrombin (20 nM) triggered virtually no responses in endothelial cells lacking PAR1 and PAR4 (bottom panels). (B) (top) Radiometric images of a single culture responding to sequential addition of PAR1 agonist (TFLLRN; 10 μM), PAR4 agonist (AYPGKF; 500 μM), and VEGF (50 ng/mL). Snapshots 1, 2, 3, and 4 were acquired at the time of peak responses for each agonist, as indicated in the bottom panel (white arrows). Par1–/– cells did not respond to PAR1 agonist (TFLLRN; 10 μM), and Par4–/– cells did not respond to PAR4 agonist (AYPGKF; 500 μM) (not shown). Note that 20% to 30% of the wild-type endothelial cells that responded to TFLLRN responded to subsequent stimulation with AYPGKF. (bottom) Fura-2 fluorescence ratios as a function of time for 3 individual cells (cells a, b, and c, indicated in the top panels). Note that each individual cell responded to all 3 agonists.