Hypoxia-induced activation of AMPK is Ca²⁺/CaMKKβ dependent. (A) ATII cells were exposed to 21% (N) or 1.5% (H) O₂ for 10 min in the presence or absence of STO-609 (20 μM, 30-min preincubation). The graph represents activation of AMPK measured as the ratio of AMPK phosphorylation at Thr-172 (pAMPKα) and total AMPKα by Western blot analysis. Values are expressed as means ± SEM (n = 4). **, P < 0.01. Representative Western blots for phosphorylation of AMPK and total AMPK protein levels are shown. (B) ATII cells were exposed to 21 or 1.5% O₂ for 60 min in the presence or absence of STO-609. The Na,K-ATPase α1 subunit (NKA-α1) plasma membrane abundance was determined by cell surface biotinylation followed by streptavidin pulldown and Western blot analysis using specific antibodies. Representative Western blots of the NKA-α1 at the plasma membrane and E-cadherin as a loading control are shown. Results are means ± SEM (n = 4). **, P < 0.01. (C) Western blot showing the expression of LKB1 in alveolar epithelial cells. (D to F) A549 p-Babe (D) A549+LKB1 (E), and A549+LKB1 KD (F) cells were transfected with siRNA against CaMKKβ or scrambled siRNA, and 48 h later cells were exposed to 21 or 1.5% O₂ for 10 min. pAMPKα and total AMPKα were determined by Western blotting. β-Actin was used as a loading control. (G) A549+LKB1 cells transfected with CaMKKβ siRNA were exposed to hypoxia, and Na,K-ATPase protein expression was assessed as for panel B. Values are expressed as means ± SEM (n = 4). **, P < 0.01. (H and I) AMP/ATP (H) and ADP/ATP (I) ratios in ATII cells exposed to 1.5% O₂ for 0, 5, and 30 min were assessed by HPLC (n = 6).

Expression of STIM1 D76A leads to AMPK activation and Na,K-ATPase downregulation. (A) A549 cells were transiently transfected with WT STIM1-YFP (WT), STIM1-YFP D76A (D76A), or empty vector (EV) and 24 h later pAMPKα, AMPKα, pACC, ACC, pPKCζ, pPKCζ, STIM1, and β-actin were determined by Western blotting. Representative Western blots are shown. (B and C) A549 cells (B) or ρ⁰-A549 cells (C) were transiently transfected with WT STIM1-YFP, STIM1-YFP D76A, or empty vector, and 24 h later cells were exposed to 21% (N) or 1.5% (H) O₂ for 60 min or treated with thapsigargin (TG) (1 μM). Na,K-ATPase α1 subunit plasma membrane abundance (NKA-α1), E-cadherin, and STIM1 were determined by Western blotting as described in the text. Values are expressed as means ± SEM (n = 5). **, P < 0.01. Representative Western blots are shown.

STIM1 mediates the hypoxia-induced impairment of AFR in rat lungs. (A) Isolated lungs from rats instilled with empty vector (EV) or shRNA-STIM1 were perfused for 1 h with 21% O₂ (N) and then switched to approximately 1.5% O₂ (H), and AFR was measured as described in the text. Bars represent means ± SEM (n = 3). **, P < 0.01. (B) Representative Western blots of STIM1, β-actin, and m-Cherry in peripheral lung tissue homogenates from mock-, EV- and shRNA-STIM1-instilled rats.

CRAC channel activity is necessary for hypoxia-induced AMPK activation and Na,K-ATPase downregulation in alveolar epithelial cells. (A) ATII cells were exposed to 21% (N) or 1.5% (H) O₂ for 10 min in the presence or absence of BAPTA-AM (20 μM, 15-min preincubation), and pAMPKα and total AMPKα were determined. Representative Western blots are shown (n = 4). (B and C) Representative traces of [Ca²⁺]_i in ATII cells exposed to hypoxia or nifedipine plus hypoxia are illustrated. Fura2-loaded cells were initially perfused with medium containing 21% O₂, and then perfusion was switched to 1.5% O₂ in absence (B) or in the presence (C) of nifedipine (5 μM, 10-min preincubation). Results are for 7 experiments with 10 to 30 cells each. (D to F) Calcium influx into ATII cells exposed to hypoxia, treated with thapsigargin (TG) (1 μM), or pretreated with LaCl₃ (5 μM, 5-min preincubation) and then exposed to hypoxia. Perfusion was started in Ca²⁺-free medium and switched to 2 mM Ca²⁺ as indicated. Results are for 5 experiments with 10 to 30 cells each. (G to I) pAMPKα and total AMPKα were determined in ATII cells preincubated with LaCl₃ and then exposed to 1.5% O₂, incubated in the presence or absence of 2 mM Ca²⁺, and exposed to 21% (N) or 1.5% (H) O₂ for 10 min or treated with vehicle (V) or TG for 10 min. Representative Western blots are shown (n = 3). (J) ATII cells were exposed to 21% (N) or 1.5% (H) O₂ for 60 min in the presence or absence of LaCl₃. The Na,K-ATPase-α1 subunit plasma membrane abundance was determined. Representative Western blots of Na,K-ATPase α1 (NKA-α1) at the plasma membrane and E-cadherin are shown. Results are means ± SEM (n = 4). **, P < 0.01.

Hypoxia-induced STIM1 redistribution results in CRAC channel activity following AMPK activation and Na,K-ATPase downregulation. (A) A549 cells were transiently transfected with full-length WT STIM1-YFP and 24 h later were exposed 21% (normoxia) or 1.5% (hypoxia) O₂, and the WT STIM1-YFP distribution was assessed. (B to D) Measurement of calcium in A549 cells transfected with siRNA against STIM1 (B), against Orai1 (C), or scrambled (D) and then exposed to hypoxia. Perfusion was started in Ca²⁺-free medium and switched to 2 mM Ca²⁺ as indicated. Results are from 4 experiments with 20 to 35 cells each. (E) Representative Western blot of the expression levels of STIM1 and Orai1 in A549 cells transfected with the respective siRNA. β-Actin was used as a loading control. (F and G) A549 cells were transfected with siRNA against STIM1 or Orai1 or with scrambled siRNA, and 48 h later cells were exposed to 21 or 1.5% O₂ for 10 min. pAMPKα, AMPKα, pACC, pPKCζ, STIM1, and Orai1 protein levels are shown. (H and I) Cells transfected as for panel E were exposed to 21 or 1.5% O₂ for 60 min, and the Na,K-ATPase α1 subunit plasma membrane (NKA-α1) abundance was determined. Representative Western blots of the NKA-α1 at the plasma membrane and in total cell lysates and STIM1 or Orai1 are shown. Results are means ± SEM (n = 4). **, P < 0.01.

Mitochondrial ROS mediate the hypoxia-induced increase in [Ca₂₊]_i. (A and B) ATII cells were treated with 100 μM t-H₂O₂ or vehicle for 10 min in the presence or absence of BAPTA-AM (20 μM, 15-min preincubation) (A) or STO-609 (20 μM, 30-min preincubation) (B), and pAMPKα and total AMPKα were determined by Western blot analysis. Representative Western blots are shown. (C) ATII cells were exposed to 21% (normoxia) or 1.5% (hypoxia) O₂ for 10 min in the presence or absence of EUK-134 (20 μM, 15-min preincubation) or were treated with t-H₂O₂ (100 μM, 10 min). Endogenous STIM1 was detected by immunofluorescence microscopy using an anti-STIM1 antibody. (D to F) Calcium influx into ρ⁰-A549 cells exposed to hypoxia, t-H₂O₂, or thapsigargin (TG). Perfusion was started in Ca²⁺-free medium and switched to 2 mM Ca²⁺ as indicated. Results are from 5 experiments with 17 to 43 cells each.

ROS generated at mitochondrial complex III are required for the hypoxia-induced increase in [Ca²⁺]_i and AMPK activation. A549 cells were infected with two different shRNAs against Rieske F-S (an Fe protein). (A and B) Calcium influx profiles for cells transfected with Fe-S shRNA1 or shRNA2 and exposed to hypoxia. Results are for 8 experiments with 7 to 15 cells each. (C and D) Profiles for cells treated with 100 μM t-H₂O₂. Results are for 5 experiments with 7 to 15 cells each. Perfusion was started in Ca2⁺-free medium and switched to 2 mM Ca²⁺ as indicated. (E and F) pAMPKα and total AMPKα were determined in cells exposed to 21% (N) or 1.5% (H) O₂ (E) or treated with t-H₂O₂ 100 μM (F). Representative Western blots for pAMPKα, AMPKα, and the Rieske Fe-S protein are shown (n = 3).