Figure 5.

ATP released from RBCs contributes to SS-induced increases in EC [Ca²⁺]_i in individually perfused venules. (A) A representative experiment showing shear-dependent increases in EC [Ca²⁺]_i when the vessel was perfused with RBC solution (40% RBCs in albumin Ringer solution), and pre-treatment of RBCs with a pannexin 1 inhibitor, CBX (100 µM) abolished such effect. (B) Summary results of SS induced release of ATP from RBCs or platelets. RBCs released ATP in a shear-dependent manner, and CBX treatment abolished SS-induced ATP release from RBCs (n = 5 per group). ATP measured in platelet rich plasma (PRP) showed no difference from platelet free plasma (PFP) and no responses to applied SS (n = 3 per group). (C) A representative experiment showing that the applied ATP (10 μM) induced a transient increase in EC [Ca²⁺]_i, and the presence of CBX (100 µM) in the perfusate did not affect baseline and ATP induced increases in EC [Ca²⁺]_i. (D) Superimposed two individual experiments showing W146 (10 μM), a selective S1P_R1 antagonist, had no effect on baseline and SS-induced increases in EC [Ca²⁺]_i in RBC perfused vessels, but abolished S1P-induced increases in EC [Ca²⁺]_i in BSA-Ringer perfused vessels. The S1P effect was recovered after removal of W146 with 40 min perfusion of albumin-Ringer. (E) Summary results of EC [Ca²⁺]_i measurements (n = 4 or 5 per group). ***P < 0.0005, **P < 0.005, NS: P > 0.05. Solid line link: one-way ANOVA with post-hoc Tukey for perfusion groups with the same SS, and unpaired student’s t-test for two group comparison. Dotted line link: paired student’s t-test for paired studies conducted in each vessel or animal.