cell culture primary human pasmcs hpasmcs  (PromoCell)

Journal: Cardiovascular Research

Article Title: Pannexin 1: a novel regulator of acute hypoxic pulmonary vasoconstriction

doi: 10.1093/cvr/cvab326

Figure Lengend Snippet: Smooth muscle Panx1 is required for the intact HPV response. Representative western blot (A) and quantitative densitometric analysis (B) show expression of Panx1 in human pulmonary artery smooth muscle cells (hPASMCs) and human pulmonary artery endothelial cells (hPAECs). GAPDH served as loading control (n = 3 per group). Panx1 expression on hPASMC and hPAEC were assessed on the same gel. Interspersed non-relevant lanes were removed and relevant sections were re-composed at the dotted line. Representative tracing (C) of pulmonary artery pressure (PAP) in isolated perfused mouse lungs and quantitative group data analysis (D) show attenuation of the vasoconstrictive response to hypoxia (1% O2)—measured as increase in PAP (ΔPAP)—by probenecid (50 mg/kg bw) (control n = 6, probenecid n = 3). (E) The pannexin 1 (Panx1) specific inhibitory peptide (10Panx1; 800 µMol/L) reduces the ΔPAP response to hypoxia by more than 50% as compared to scrambled peptide control (scrambled peptide n = 5, 10Panx1 n = 4). (F) Pulmonary vasoconstriction in response to KCl was attenuated by 10Panx1 (800 µMol/L) (KCl n = 4, KCl+10Panx1 n = 3). (G) Tamoxifen induced deletion of Panx1 in smooth muscle cells (SMMHC-CreERT2/Panx1fl/fl), yet not Panx1 deletion in endothelial cells (Cdh5-CreERT2/Panx1fl/fl) attenuates ΔPAP response to hypoxia in isolated perfused murine lungs (n = 4–5 per group). (I and J) Hypoxia did neither change Panx1 RNA levels (n = 4 per group) nor Panx1 protein expression (n = 3 per group). Data are mean ± SEM; Data were analysed using Mann–Whitney U-test (A–F, H–J) or Kruskal–Wallis test (Figure test); *P < 0.05; ns, not significant.

Article Snippet: 2.4 Cell culture Primary human PASMCs (hPASMCs) and primary human pulmonary artery endothelial cells (hPAECs) from five different male caucasian donors were purchased from PromoCell (Heidelberg, Germany) and cultured at 95% relative humidity, 37°C and 5% CO 2 using Smooth Muscle Cell Growth Medium (PromoCell; Heidelberg, Germany) and Endothelial Cell Growth Medium MV2 (PromoCell; Heidelberg, Germany), respectively, containing 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin.

Techniques: Western Blot, Expressing, Isolation, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Pannexin 1: a novel regulator of acute hypoxic pulmonary vasoconstriction

doi: 10.1093/cvr/cvab326

Figure Lengend Snippet: Panx1 does not regulate HPV via ATP release and purinergic signalling. (A) Group data show ATP concentration in supernatant and cytosolic fraction of hPASMCs under normoxic (21% O2; white bars) and hypoxic (1% O2; grey bars) conditions after 5 min. Hypoxia decreased both extracellular and intracellular ATP levels (n = 3 per group). (B) Longitudinal measurements over 15 min of hypoxia show a corresponding decrease in extracellular ATP concentration. (C) In isolated perfused mouse lungs, pretreatment with the ATP degrading enzyme apyrase (8 U/mL and 16 U/mL) did not alter the vasoconstrictive response to hypoxia (1% O2), measured as increase in pulmonary artery pressure (ΔPAP) (control n = 5, apyrase 8 U/mL n = 3, apyrase 16 U/mL n = 3). (I) In isolated perfused mouse lungs, administration of an ATP bolus (0.9 µmol) induced pulmonary vasoconstriction (n = 6), measured as increase in pulmonary artery pressure (ΔPAP), which was reduced by apyrase (8 U/mL; n = 5). (J) In isolated perfused mouse lungs, pretreatment with the purinergic receptor blocker suramin (100 µMol/L) did not alter the vasoconstrictive response to hypoxia (control n = 5, suramin n = 3). (K) Suramin (100 µMol/L) attenuated the vasoconstrictive response to an ATP bolus (0.9 µmol) (ATP = 6, ATP + suramin n = 5). Data are mean ± SEM; data were analysed using Mann–Whitney U-test (A, D–F) or Kruskal–Wallis test (C); *P < 0.05; ns, not significant.

Article Snippet: 2.4 Cell culture Primary human PASMCs (hPASMCs) and primary human pulmonary artery endothelial cells (hPAECs) from five different male caucasian donors were purchased from PromoCell (Heidelberg, Germany) and cultured at 95% relative humidity, 37°C and 5% CO 2 using Smooth Muscle Cell Growth Medium (PromoCell; Heidelberg, Germany) and Endothelial Cell Growth Medium MV2 (PromoCell; Heidelberg, Germany), respectively, containing 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin.

Techniques: Concentration Assay, Isolation, MANN-WHITNEY