Fig. 7. Activation of the P2X7 receptor on APCs by 3 μM ATP initiates the heightened immune responses associated with hypertension.

(A) Splenocytes from either C57BL/6 or P2X7 knockout mice were labeled with the Ca²⁺ probe Fluo-4 AM in vitro. The cells were then treated with either 3 or 500 μM ATP, and Ca²⁺ influx into DCs (left) and T cells (middle) was monitored continuously for 4 min by flow cytometry (right). The first minute kinetics of Ca²⁺ influx into DCs in the presence of 3 μM ATP is also shown. (B) Splenic DCs were stimulated with or without 3 μM ATP. Some of the ATP-treated cells were pretreated with the calcium chelator BAPTA or the calmodulin inhibitor chlorpromazine (Chlor). Surface CD86 was measured after 18 hours. (C) Peritoneal macrophages were treated with 3 or 500 μM ATP for 18 hours ex vivo, and IL-1β in the medium was measured by ELISA. Data were analyzed with paired Student′s t test. (D) Splenic DCs and T cells were purified from the spleen of naїve mice. DCs (2 × 10⁶), T cells, and HEK-293 cells were pelleted and then dissolved in 100 μl of acetonitrile. The concentrations of LPCs C16:0 and C18:0 were determined by LC-MS/MS. (E) Concentrations of LPCs C16:0 and C18:0 in the plasma of normotensive and hypertensive mice. (F) Splenic DCs from normotensive and hypertensive mice were treated with vehicle, apyrase, or A740003 for 18 hours. Surface expression of CD86 was then measured. (G) Normotensive and hypertensive mice were pretreated intraperitoneally with apyrase (0.2 U/g weight) or A740003 (50 nmol/mouse) every other day for 6 days, and they were then immunized with OVA-CFA. Apyrase or A740003 was continued after immunization. The percentages of OVA-specific CD8⁺ T cells in the blood were evaluated 7 days after immunization. (H and I) Normotensive and hypertensive mice were treated with ConA to induce hepatitis with or without the P2X7 receptor antagonist A740003. Plasma ALT levels (H) and CD4⁺ T cell cytokines (I) were measured.