ATP secretion after hypotonic shock and acute effect on <i>I</i>_<i>sc</i> generated by alveolar epithelial cells.

<p>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 (ATP_ap) or bilateral, but not basolateral (ATP_ba) addition of ATP, decreased total transepithelial current generated by alveolar epithelial cells (<b>B</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 (<b>C</b>). 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 (<b>D</b>). Apyrase treatment had no significant effect on the <i>I</i>_<i>sc</i> 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.</p

Kinetic of [Ca²⁺]_i concentration rise after 20% hypotonic shock in alveolar epithelial cells.

<p>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 <b>A</b>. Hypotonic shock induced an immediate increment of intracellular [Ca²⁺]_i, reaching maximum after 266.3 ± 27 s (T_max) compared to the basal condition (T₀). The F₃₄₀/F₃₈₀ ratio rose significantly between T₀ and T_max (N=4, *p<0.05 by Mann-Whitney Test) (<b>B</b>). The time to reach maximum <i>I</i>_<i>sc</i> and maximum [Ca²⁺]_i was significantly different (N=17 for <i>I</i>_<i>sc</i>, N=5 for F₃₄₀/F₃₈₀, *p<0.05 by Mann-Whitney Test) (<b>C</b>).</p

Implication of Cl^- channels and KCC in basal and hypotonic-induced transepithelial current.

<p>Basal transepithelial current (<i>I</i>_<i>sc</i> Basal), hypotonic-induced current (<i>I</i>_<i>sc</i> 20% Hypo) and the current rise elicited by hypotonic shock (Δ <i>I</i>_<i>sc</i> Shock) are depicted in the basal conditions (Ctrl), after treatment with 100 µM basolateral bumetanide (Bumet), 100 µM apical (NPPB_a) or basolateral (NPPB_b) 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 <i>I</i>_<i>sc</i>, 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 <i>I</i>_<i>sc</i> Basal. N≥4, *p<0.05 by Mann-Whitney Test compared to untreated controls. # p<0.05 by Mann-Whitney Test compared to NPPB_a, ¤ p<0.05 by Mann-Whitney Test compared to NPPB_b or DIOA.</p

Inhibition of calmodulin kinase by W7 decreases the hypotonic shock current rise.

<p>Alveolar epithelial cells were pretreated for 20 min with 25 µM W7, a Ca²⁺/calmodulin antagonist, before <i>I</i>_<i>sc</i> recording in Ussing chamber. Total (<i>I</i>_<i>sc</i> 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 <i>I</i>_<i>sc</i> rise after 20% hypotonic shock. N≥4, *p<0.05 by Mann-Whitney Test compared to Basal.</p

Channels and transporters involved in ionic transport in basal and hypotonic shock conditions.

<p>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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074565#B59" target="_blank">59</a>], 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; <b>T</b> : Inhibitor.</p

Role of Na⁺ and K⁺ currents in basal and hypotonic-induced transepithelial current.

<p>Basal transepithelial current (<i>I</i>_<i>sc</i> Basal), hypotonic-induced current (<i>I</i>_<i>sc</i> 20% Hypo) and the current rise elicited by hypotonic shock (Δ <i>I</i>_<i>sc</i> 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 <i>I</i>_<i>sc</i>, 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.</p

Supplementary Material for: Thrombolysis for acute wake-up and unclear onset strokes with alteplase at 0.6 mg/kg in clinical practice: THAWS2 Study

Introduction: The aim of this study was to determine the safety and efficacy of intravenous (IV) alteplase at 0.6 mg/kg for patients with acute wake-up or unclear onset strokes in clinical practice. Methods: This multicenter observational study enrolled acute ischemic stroke patients with last-known-well time >4.5 h who had mismatch between DWI and FLAIR and were treated with IV alteplase. The safety outcomes were symptomatic intracranial hemorrhage (sICH) after thrombolysis, all-cause deaths and all adverse events. The efficacy outcomes were favorable outcome defined as an mRS score of 0–1 or recovery to the same mRS score as the premorbid score, complete independence defined as an mRS score of 0–1 at 90 days, and change in NIHSS at 24 h from baseline. Results: Sixty-six patients (35 females; mean age, 74±11 years; premorbid complete independence, 54 [82%]; median NIHSS on admission, 11) were enrolled at 15 hospitals. Two patients (3%) had sICH. Median NIHSS changed from 11 (IQR, 6.75–16.25) at baseline to 5 (3–12.25) at 24 h after alteplase initiation (change, –4.8±8.1). At discharge, 31 patients (47%) had favorable outcome and 29 (44%) had complete independence. None died within 90 days. Twenty-three (35%) also underwent mechanical thrombectomy (no sICH, NIHSS change of –8.5±7.3), of whom 11 (48%) were completely independent at discharge. Conclusions: In real-world clinical practice, IV alteplase for unclear onset stroke patients with DWI-FLAIR mismatch provided safe and efficacious outcomes comparable to those in previous trials. Additional mechanical thrombectomy was performed safely in them