Figure 5.

Alpha-1 antitrypsin (AAT) signaling involves nicotinic acetylcholine receptors (nAChR) and inhibits P2X7 receptor function. Lipopolysaccharide (LPS)-primed U937 cells were stimulated with 2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate (BzATP) in the presence or absence of Prolastin^® (AAT-P, 1 mg/ml). (A–C) Interleukin (IL)-1β released to the supernatant was measured after 30 min. (A) Nicotinic antagonists mecamylamine (Mec), α-bungarotoxin (α-Bun), strychnine (Stry), RgIA4, or ArIB [V11L, V16D] were added together with BzATP and AAT-P, *p ≤ 0.05 compared to cells treated with LPS, BzATP, and AAT-P. (B,C) Expression of nAChR subunits α7, α9, and α10 was silenced by siRNA (si α7, α9, α10). Cells were either transfected with a single siRNA (B) or with a combination of two of them (C). In addition, cells were transfected with control siRNA (si con); *p ≤ 0.05, compared to data from respective experiments on cells treated with si con. (C). (D,E) Whole-cell patch-clamp experiments were performed on LPS-primed U937 cells stimulated with BzATP. Patch-clamp recordings of U937 cells are depicted in (D), and changes in ion currents (ΔI) are summarized in (E). BzATP was applied twice (I and II). The second BzATP stimulus was given in the presence or absence of AAT-P and Mec. Data are presented as individual data points, bar represents median, whiskers encompass the 25th to 75th percentile, n-numbers of independent experiments are indicated in the figure. Experimental groups were compared by Wilcoxon signed-rank test (E) or by Kruskal–Wallis followed by Mann–Whitney rank sum test (A–C).