Fig 1.
TASK-1 Knockout (KO) mice and expression of TASK-1 and TASK-3 mRNA in murine organs.
(A) Genotype identification of TASK-1 wild-type (WT), heterozygous (HZ), and KO mice by PCR, agarose gel. (B) Expression of TASK-1 and TASK-3 mRNA in WT murine organs, RT-PCR, agarose gel. ØRT is lung sample processed without RT. β-Actin was used as internal control. Heart served as positive control. H2O is control without cDNA template.
Fig 2.
Laser-assisted Microdissection (LMD) and expression of TASK-1 in WT murine intra-pulmonary arteries.
(A) LMD. The white line indicates the luminal diameter of a pre-acinar artery. It represents the maximum distance between intimal surfaces at right angle (90°) to the longitudinal axis. The area of vessel to be picked was first marked (blue lines; left panel). The marked piece of tissue (missing in right panel) was catapulted into the lid of a reaction tube and processed for RT-PCR. (B) RT-PCR of laser-assisted microdissected samples, agarose gel. ØRT is sample of cardiomyocytes processed without Reverse Transcriptase (RT). β-Actin was used as internal control. Heart served as positive control. H2O is control without cDNA template.
Fig 3.
One-Dimensional (1-D) and Two-Dimensional (2-D) gel electrophoresis followed by anti-TASK-1 western blotting.
(A) 1-D Gel electrophoresis followed by western blotting with anti-TASK-1 antibody lot AN 02. Immunoreactivity was prominent in the cerebellum and heart and was weak in lung extracts of TASK-1 wild-type (WT), heterozygous (HZ), and knockout (KO) mice. (B) 1-D Gel electrophoresis followed by western blotting with anti-TASK-1 antibody lot AN 08. Labelling with anti-TASK-1 antibody was detectable in lung and heart extracts of WT and KO animals, but was absent in the cerebellum of both mouse strains. Staining was absent in control without primary antibody. (C) 2-D Gel electrophoresis of the cerebellum of WT and TASK-1 KO mice followed by anti-TASK-1 western blotting. Comparable labelling patterns were present in samples of both mouse strains with anti-TASK-1 antibody lot AN 02. Immunoreactive spots were identified as glial fibrillary acidic protein isoform (GFAP2), and mitochondrial cytochrome b-c1 complex subunit 1 (cyt b-c1). Molecular weights (kDa) are presented on the left side of blots. The solid lines indicate assembled blots.
Fig 4.
Videomorphometric analysis of the hypoxic response of intra- (A) and pre-acinar (B) arteries of WT and TASK-1 KO mice.
The luminal area of vessels before exposure to hypoxia was set as 100%, and changes in the luminal areas are presented as relative values. Hypoxia-induced constriction of intra-acinar arteries was comparable in both mouse strains, whereas in KO pre-acinar arteries, it started to fade earlier as compared to WT. Data are presented as mean ± SEM. n is number of vessels/number of animals. Tests of significance were done at the time points indicated by arrows. *, #, §, $ P≤ 0.05 and **, ## P≤ 0.01 (Mann–Whitney U-test).
Fig 5.
Impact of TASK-1 inhibitor anandamide on vasoconstriction of WT mice.
(A) Effect of anandamide on intra-acinar arteries. Anandamide induced constriction at normoxia and reduced hypoxia-induced vasoconstriction significantly at one time. (B) Effect of anandamide on pre-acinar arteries. Anandamide did not induce constriction at normoxia. However, it reduced hypoxia-induced vasoconstriction significantly after 20 min. Data are presented as mean ± SEM. n is number of vessels/number of animals. Tests of significance were done at the time points indicated by arrows. *, #, §, $ P≤ 0.05 and **, ## P≤ 0.01 (Mann–Whitney U-test).
Fig 6.
Impact of TASK-1 inhibitor A293 on vasoconstriction of WT and TASK-1 KO mice.
A293 induced constriction of WT intra- (A) and pre-acinar (B) arteries at normoxia and reduced hypoxia-induced vasoconstriction. In KO intra- (C) and pre-acinar (D) arteries at normoxia, the constrictory effect of A293 persisted. Data are presented as mean ± SEM. n is number of vessels/number of animals. Tests of significance were done at the time points indicated by arrows. *, #, §, $ P≤ 0.05 and **, ##, §§, $ $ P≤ 0.01 (Mann–Whitney U-test).
Fig 7.
Hypoxia-induced changes in Pulmonary Arterial Pressure (ΔPAP) of isolated, perfused, and ventilated lungs of WT and TASK-1 KO mice.
ΔPAP was referenced to the onset of 180 min of hypoxic ventilation period (time set at 0). Normoxic PAP and its increase during hypoxic ventilation were neither different between KO and in WT mice (A) nor when comparing WT mice in the presence or absence of the TASK-1 inhibitor A293 (B). Data are presented as mean ± SEM. n refers to number of animals used. HPV: hypoxic pulmonary vasoconstriction.