Journal: Laryngoscope investigative otolaryngology

Article Title: Development of a physiological model of human middle ear epithelium.

doi: 10.1002/lio2.661

Figure Lengend Snippet: FIGURE 5 (A) Human middle ear epithelia show ENaC and CFTR activity. Mean short circuit current (Isc) measurements over time from two middle ear epithelial transwells run in duplicate. ENaC activity was measured as a change in Isc (Δisc) induced by amiloride (Amil, 10 μM, apical). CFTR activity was measured as change in Isc (ΔIsc) induced by forskolin (FSK, 10 μM, bilateral) and CFTRinh172 (Inh- 172, 20 μM, apical). (B) Absolute mean lsc with the addition of Amil, FSK, and Inh-172

Article Snippet: The membrane-bound epithelial cells were inserted into the EasyMount Ussing chamber system and bathed in bilateral modified bicarbonate Kreb's solution, continuously gassed with 95% O2/5% CO2 maintained at 37 C. Cells were voltage-clamped to 0 mV and left to equilibrate before adding the inhibitors and agonists, which were added in the following order: amiloride (10 μM apical; Sigma-Aldrich), forskolin (10 μM, bilateral; Tocris Bioscience, Bristol, UK), CFTRinh172 (20 μM, apical; Tocris Bioscience).

Techniques: Activity Assay

Journal: Journal of Clinical Medicine

Article Title: Comparison of the Effect of CFTR Modulators elexacaftor / tezacaftor / ivacaftor and lumacaftor / ivacaftor via Serum Human Epididymis Protein 4 Concentration in p.Phe508del-CFTR Homozygous Cystic Fibrosis Patients

doi: 10.3390/jcm14176188

Figure Lengend Snippet: Rescue of functional p.Phe508del-CFTR Cl− currents in Kaftrio ® - or Orkambi ® -treated CFBE 41o- cells. Representative whole-cell Cl − current traces were elicited by voltage steps from a holding potential of −40 mV to a series of test potentials ranging from 0 to +60 mV in 20 mV increments every 10 s in CFBE 41o- cells expressing wt-CFTR (column A ) or p.Phe508del-CFTR (column B ) or in cells expressing deletion mutant CFTR but treated for 24 h with LUM/IVA (column C ) or ELX/TEZ/IVA (column D ) for 24 h. The duration of the depolarizing pulses was 1 s. For clarity, Cl − currents recorded at 0 mV are shown throughout the figure. Top panels : basal currents recorded at 0 mV in the absence of FSK/IBMX stimulation (black), recorded upon ∼2 min stimulation by FSK/IBMX (10/100 μM) (blue) and in the presence of FSK/IBMX and 20 μM of CFTR inh172 (red). The middle panels show the corresponding peak current densities determined at 0 mV (pA/pF, mean ± SEM) obtained in the absence of FSK/IBMX (basal, black symbols), upon stimulation by FSK/IBMX (blue symbols), and in the presence of FSK/IBMX and 20 μM CFTR inh172 (red symbols). The current density for a particular cell was determined as the average of current density values obtained for three to four consecutive depolarizing pulses repeated every 5 s. Bottom panels: current density–voltage relationships (pA/pF, mean ± SEM) measured at the indicated test potentials in the absence of FSK/IBMX (basal, black symbols), upon stimulation by FSK/IBMX (blue symbols), and in the presence of FSK/IBMX and 20 μM CFTR inh172 (red symbols). ( E ) Analysis of the basal peak current densities (pA/pF) determined at +40 mV and recorded in cells in the absence of CFTRm pretreatment (F580del, empty circles), pre-treated with LUM/IVA (up triangles) or ELX/TEZ/IVA (down triangles). ( F ) Analysis of the FSK (10 µM)/IBMX (100 µM)-stimulated peak current densities (pA/pF) recorded in cells in the absence of CFTRm pretreatment ( p.Phe508del-CFTR , empty circles), pre-treated with LUM/IVA (up triangles) or ELX/TEZ/IVA (down triangles). ( G ) Analysis of the FSK/IBMX-stimulated and CFTR inh172 (20 µM)-inhibited peak current densities (pA/pF) recorded in cells in the absence of CFTRm pretreatment (F580del, empty circles), pre-treated with LUM/IVA (up triangles) or ELX/TEZ/IVA (down triangles). Data are expressed as mean ± SEM (n = 7–16 cells/condition) unless otherwise indicated, and each symbol represents an individual record. An unpaired or paired t-test was performed for comparisons. Differences were considered significant at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Article Snippet: CFTR correctors elexacaftor (VX-445) (S8851), lumacaftor (VX-809) (S1565), and tezacaftor (VX-661) (S7059); CFTR potentiator ivacaftor (VX-770) (S1144); voltage-independent selective CFTR inhibitor CFTR inh172 (S7139); and CFTR activator (FSK, S2449) were purchased from Selleck Chemicals (Houston, TX, USA). cAMP phosphodiesterase inhibitor IBMX (I5879) was ordered from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Functional Assay, Expressing, Mutagenesis