Figure 1.
LXA4 effect on intracellular Ca2+ activity in non-CF and CF bronchial epithelial cells.
(A) Typical effect of LXA4 (100 nM) on the cytosolic Ca2+ (ratio F340/F380) measured in Nuli-1 and CuFi-3 cell lines in control (plain line) and in external Ca2+ -free conditions (dotted line). (B) Typical effect of LXA4 (100 nM) on the cytosolic Ca2+ in normal and CF primary cultures of bronchial epithelial cells (NHBE and CFBE). (C) Mean values of the maximum increase in Ca2+ (peak) and measured 2 and 5 minutes after the peak, in Nuli-1 (n = 6), CuFi-1 (n = 6), CuFi-3 (n = 6), CuFi-4 (n = 4) cell lines and in NHBE (n = 4) and CFBE (n = 4) bronchial epithelial cells in primary culture (* p<0.05, ** p<0.01).
Figure 2.
Effect of Boc-2 on the intracellular Ca2+ signal induced by LXA4.
(A) Representative effect of LXA4 (100 nM) and ATP (100 µM) on cytosolic Ca2+ (ratio F340/F380) in NuLi-1 cells in control conditions (upper panel) and after 24 hours of pre-treatment with Boc-2 (10 µM) a specific inhibitor of ALX/FPR2 (lower panel). (B) Mean values corresponding to the effect of Boc-2 on the Ca2+ response to LXA4 in Nuli-1 (n = 5) and in CuFi-3 (n = 4) cell lines (*** p<0.001).
Figure 3.
Time dependency of the effect of LXA4 on whole-cell currents in normal and CF bronchial epithelial cells in primary culture.
Typical I-V relationships and corresponding current records obtained before and after 5, 10 and 15 min exposure to LXA4 (100 nM) in NHBE (A) and CFBE (B) isolated bronchial epithelial cells in primary culture. (C) Mean values corresponding to the time dependency of the LXA4 effect onwhole cell inward conductances in Nuli-1 (n = 8) and in CuFi-3 (n = 4) cell lines and NHBE (n = 8) and CFBE (n = 6) primary cultures (**p<0.01).
Figure 4.
Dose dependency of the effect of LXA4 on whole-cell currents of normal (Nuli-1) and CF (CuFi-3) bronchial epithelial cell lines.
Typical I-V relationships obtained before and after 10 min exposure to 1 pM (n = 6 Nuli-1, n = 6 CuFi-3), 1 nM (n = 4 Nuli-1, n = 4 CuFi-3), 10 nM (n = 3 Nuli-1, n = 3 CuFi-3) and 100 nM (n = 6 Nuli-1, n = 6 CuFi-3) LXA4 in Nuli-1 (A) and CuFi-3 (B) cell lines. (C) Mean inward conductance changes normalized to control values (gi/gic obtained without LXA4) as a function of LXA4 concentration in Nuli-1 (open bars) and CuFi-3 (black bars) cells in control conditions and obtained upon exposure to Boc-2 (10 µM) alone (10 min, 100 nM, n = 4 Nuli-1, n = 4 CuFi-3) or with Boc-2 (10 µM) and LXA4 (10 min, 100 nM, n = 6 Nuli-1, n = 6 CuFi-3) and after BAPTA-AM pre-treatment alone (n = 6 Nuli-1, n = 6 CuFi-3) or with BAPTA-AM and LXA4 (10 min, 100 nM, n = 4 Nuli-1, n = 6 CuFi-3).
Table 1.
Mean outward (Gout) and inward (Gin) conductances and reversal potentials (Erev) measured in non CF and CF airway epithelial cells in primary culture and NuLi-1 and CuFi-3 cell lines in control conditions and stimulated by LXA4 (100 nM, 10 min).
Figure 5.
Effects of Cl− channel inhibitors on whole-cell currents stimulated by LXA4.
(A) Representative I-V relationship of the effect of NPPB (1 µM) after the stimulation of primary cultures of bronchial epithelial cells with LXA4 (100 nM). Corresponding histogram of the inhibitory effect of NPPB on the inward conductance obtained in non-CF primary NHBE cells (n = 4), CuFi-3 cells (n = 3) and CF primary CFBE cells (n = 3) after stimulation with LXA4 (100 nM, 10 min). (B). Typical I-V relationships of the effect of LXA4 (100 nM) obtained in NuLi-1 cells after treatment with the CFTR channel inhibition CFTR-inh172 (5 mM) and histogram showing the absence of inhibitory effect in Nuli-1 cells (n = 3).
Figure 6.
LXA4 (100 nM) effects on airway surface liquid height in NuLi-1 (A), CuFi-3 (B) and CuFi-1 epithelial cell line (C) and CFBE primary cultures (D).
Epithelial cells were stained with calcein green, and the ASL labelled with dextran-conjugated Texas red™ fluorochrome. For each cell preparation, typical z-plane confocal sections showing ASL responses to LXA4 (top) and mean ASL height changes (bottom) in control conditions or following LXA4 (100 µM) exposure in NuLi-1,CuFi-3, CuFi-1 and CFBE epithelia treated or not with Boc-2 (10 µM) and with bumetanide (10 µM).