Figure 1.
Modulation of Isc after a 20% hypotonic shock in alveolar epithelial cells.
A typical Isc tracing is depicted in A. ENaC current was determined after adding 1 µM amiloride (Amil 1 µM) on the apical side. After apical medium perfusion (Perf) to wash away amiloride, the cells were subjected to bilateral 20% hypotonic shock (20% Hypo). Transepithelial resistance (Rte) was monitored throughout the experiment by applying a 1-mV pulse every 10 s (seen as spikes in the Isc trace). ENaC and amiloride-insensitive currents were then tested by adding 1 µM amiloride (Amil 1 µM) and 100 µM amiloride (Amil 100 µM) respectively. Total transepithelial current (Total), 1µM amiloride-sensitive ENaC current (ENaC) and the 100 µM amiloride–insensitive current (Amil-ins) are depicted in B for the untreated control condition (Basal) and after 20% hypotonic shock (20% Hypo). N≥10, *p<0.05 by Mann-Whitney Test between 20% Hypo and Basal. NS: non significant. Mean Isc from different experiments shows the transient nature of the acute rise in the transepithelial current after 20% hypotonic shock (C). Mean time to reach maximum current is depicted on top (113.6 ± 7 s; N=17). The time to return to basal values is depicted at the bottom (733.1 ± 120 s; N≥5).
Figure 2.
Impact of 20% hypotonic shock on the Ussing Isc recording.
Typical Isc tracings are depicted for current responses to 20% hypotonic shock (20% Hypo) in the basal condition (Ctrl) A, after 100 µM amiloride pre-treatment (Amiloride) B, in reduced Cl- buffer (9mM NaCl; 150 mM Na gluconate) (Cl-(-)) C or after 100 µM basolateral DIOA treatment D. Arrows show the start of hypotonic shock (20% Hypo) and the apical addition of 10 µM amiloride (Amil), 1 µM amiloride (Amil 1), 100 µM amiloride (Amil 100) or 100 µM NPPB. A double-headed arrow shows the current rise elicited by hypotonic shock (Δ Isc Shock).
Figure 3.
Role of Na+ and K+ currents in basal and hypotonic-induced transepithelial current.
Basal transepithelial current (Isc Basal), hypotonic-induced current (Isc 20% Hypo) and the current rise elicited by hypotonic shock (Δ Isc Shock) are depicted in the basal condition (Ctrl), potassium-free condition (K+(-)) or after pretreatment with 1µM amiloride (Amil 1), 100 µM amiloride (Amil 100) or 100 µM basolateral clofilium (Clofi). In experiments where the cells were pre-treated with an inhibitor that impacted on Isc, hypotonic shock was induced when the current was stabilized. For amiloride the current was stable after 5 min. In potassium-free condition (K+(-)) and clofilium pre-treatment, a longer incubation was needed (~30 min). N≥4, *p<0.05 by Mann-Whitney Test compared to untreated controls.
Figure 4.
Implication of Cl- channels and KCC in basal and hypotonic-induced transepithelial current.
Basal transepithelial current (Isc Basal), hypotonic-induced current (Isc 20% Hypo) and the current rise elicited by hypotonic shock (Δ Isc Shock) are depicted in the basal conditions (Ctrl), after treatment with 100 µM basolateral bumetanide (Bumet), 100 µM apical (NPPBa) or basolateral (NPPBb) NPPB, in bilateral Cl- reduced buffer (Cl-(-)) or 100 µM basolateral DIOA. In experiments where the cells were pre-treated with an inhibitor that impacted on Isc, hypotonic shock was induced when the current was stabilized. For apical and basolateral NPPB, a 5 to 10 min incubation was needed while the current stabilized after ~30 min for DIOA. Pre-treatment with bumetanide from 10 min to 30 min did not have an impact on Isc Basal. N≥4, *p<0.05 by Mann-Whitney Test compared to untreated controls. # p<0.05 by Mann-Whitney Test compared to NPPBa, ¤ p<0.05 by Mann-Whitney Test compared to NPPBb or DIOA.
Figure 5.
Role of apical Cl- channels and KCC co-transport in ENaC current magnitude.
ENaC current values were determined after pretreatments with 20 µM CFTR inh-172 (CFTR inh), reduced Cl- buffer (9mM NaCl; 150 mM Na gluconate) (Cl-(-)), 100 µM apical NPPB (NPPBa), 100 µM basolateral NPPB (NPPBb), or 100 µM basolateral DIOA. In experiments where the cells were pre-treated with an inhibitor that impacted on Isc, hypotonic shock was induced when the current was stabilized. For apical and basolateral NPPB, a 5 to 10 min incubation was needed while the current stabilized after ~30 min for DIOA. Pre-treatment with CFTR inh-172 and NPPBb did not have an impact on Isc ENaC. N≥5, *p<0.05 by Mann-Whitney Test compared to untreated controls (Ctrl).
Figure 6.
Impact of apical or basolateral Cl- (-) gradients on the current rise elicited by 20% hypotonic shock.
The current rise elicited by hypotonic shock (Δ Isc Shock) was tested in the basal condition (Cl-(+)), with bilateral exposure to a buffer of reduced Cl- concentration (9 mM NaCl; 150 mM Na gluconate) (Cl-(-)), or by adding this buffer at the apical side (Cl-(-) api) or basolateral side (Cl-(-) baso) only. N≥4, *p<0.05 by Mann-Whitney Test compared to Cl-(+).
Figure 7.
Kinetic of [Ca2+]i concentration rise after 20% hypotonic shock in alveolar epithelial cells.
F340/F380 ratio kinetic of the [Ca2+]i rise detected by Fura-2 Ca2+ fluorescence after 20% hypotonic shock in alveolar epithelial cells is depicted in A. Hypotonic shock induced an immediate increment of intracellular [Ca2+]i, reaching maximum after 266.3 ± 27 s (Tmax) compared to the basal condition (T0). The F340/F380 ratio rose significantly between T0 and Tmax (N=4, *p<0.05 by Mann-Whitney Test) (B). The time to reach maximum Isc and maximum [Ca2+]i was significantly different (N=17 for Isc, N=5 for F340/F380, *p<0.05 by Mann-Whitney Test) (C).
Figure 8.
Implication of Ca2+ in basal and hypotonic-induced transepithelial current.
Basal transepithelial current (Isc Basal), hypotonic-induced current (Isc 20% Hypo), the current rise elicited by hypotonic shock (Δ Isc Shock) and the amplitude of ENaC current recorded after hypotonic shock (Isc ENaC Shock) are depicted in basal condition (Ctrl), after treatment for 20 min with 50 µM BAPTA-AM (Bapta) or in a physiological buffer devoid of Ca2+ (Ca2+(-)). N≥5, *p<0.05 by Mann-Whitney Test compared to untreated controls.
Figure 9.
Inhibition of calmodulin kinase by W7 decreases the hypotonic shock current rise.
Alveolar epithelial cells were pretreated for 20 min with 25 µM W7, a Ca2+/calmodulin antagonist, before Isc recording in Ussing chamber. Total (Isc Total) and ENaC currents were evaluated in basal condition (Basal; white) or after 20% hypotonic shock (20% Hypo; gray). W7 pretreatment inhibited the total and ENaC Isc rise after 20% hypotonic shock. N≥4, *p<0.05 by Mann-Whitney Test compared to Basal.
Figure 10.
ATP secretion after hypotonic shock and acute effect on Isc generated by alveolar epithelial cells.
The time-course of 20% hypotonic shock (20% hypo) in apical ATP accumulation is depicted in A. At T0, the same volume of liquid was added to the apical and basolateral sides of the cell monolayers to achieve 20% hypotonic shock (▲) with H2O or an equivalent volume of physiological buffer (■) for the isotonic condition. Aliquots of apical medium were sampled at different time points, and ATP was measured by luciferin/luciferase assay. N≥7, *p<0.05 by Mann-Whitney Test between 20% Hypo at 1 min and time 0. Apical (ATPap) or bilateral, but not basolateral (ATPba) addition of ATP, decreased total transepithelial current generated by alveolar epithelial cells (B). N≥5, *p<0.05 by Mann-Whitney Test between apical or bilateral addition of ATP and untreated cells. NS, non significant. Bilateral addition of ATP decreased ENaC (1 µM amiloride-sensitive) current (C). N=6, *p<0.05 by Mann-Whitney Test between ATP and untreated controls (Ctrl). To rule out a role of ATP in hypotonic shock modulation of currents, the cells were pretreated with the ATP scavenger apyrase (10 U/ml; Apyr) for 2 min before challenging the monolayers with 20% hypotonic shock (D). Apyrase treatment had no significant effect on the Isc increase induced by hypotonic shock. N≥4, NS: non significant by Mann-Whitney Test between 20% Hypo in presence of absence of Apyr. *p<0.05 by Mann-Whitney Test between basal (white) or 20% hypo (grey) treated cells.
Figure 11.
Channels and transporters involved in ionic transport in basal and hypotonic shock conditions.
In the basal condition, a transcellular Cl- transport via NPPB-sensitive apical channels (Cl- ch) and basolateral K+/Cl- co-transporter (KCC) generates membrane potential that is optimal for Na+ transport via amiloride-sensitive ENaC and NSC. Although alveolar epithelial cells express CFTR [59], in the absence of a cAMP agonist, CFTR is not involved in basal Na+ transport. During a 20% hypotonic shock, Ca2+/calmodulin and a basolateral Cl- Influx play a central role in the transient current rise elicited by hypotonicity. NPPB-sensitive and insensitive pathways are involved for this flux. Although basolateral K+ channels (K+ ch) are not involved in the current rise elicited during hypotonic shock, their inhibition blunts current normalization after hypotonic shock. →: Ionic flux; T : Inhibitor.