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Revisiting CFTR inhibition: a comparative study of CFTRinh -172 and GlyH-101 inhibitors.

Melis N, Tauc M, Cougnon M, Bendahhou S, Giuliano S, Rubera I, Duranton C - Br. J. Pharmacol. (2014)

Bottom Line: We also explored the effect of both inhibitors on cell viability using live/dead and cell proliferation assays in two different cell lines.The CFTRinh -172 did not affect the CaCC but did inhibit the VSORC, at concentrations higher than 5 µM.Our results provided insights into their use in mouse models.

View Article: PubMed Central - PubMed

Affiliation: University of Nice-Sophia Antipolis, LP2M CNRS-UMR7370, Faculté de médecine, Nice, France.

No MeSH data available.


Related in: MedlinePlus

Inhibition of forskolin-activated CFTR-like conductance by CFTRinh-172 and GlyH-101 inhibitors in CFTR-expressing cells. (A and B) Whole-cell current traces recorded in CFTR-expressing cells (kidney, DCT cells) under control condition and after forskolin exposure (FK, 1–10 µM). Once the Cl− conductance is fully developed (3–4 min), CFTRinh-172 or GlyH-101 were perfused at increasing concentrations (0.5, 1, 5, 10 µM). The membrane potential was held at −40 mV and currents were elicited by a train of 11 voltage steps (400 ms duration) between −100 and +100 mV in +20 mV increment. The zero current level is indicated by a dashed line. (C) Mean current/voltage relationships measured at 350 ms after the onset pulse corresponding to experiments performed (A and B) under control condition, after FK exposure and finally in the presence of CFTRinh-172 (10 µM) or GlyH-101 (10 µM). Values are means (±SEM) of six to eight individual cells. (D and E) Histograms illustrating the concentration-dependent inhibition of the CFTR conductance obtained with increasing concentrations of CFTRinh-172 (D) or GlyH-101 (E). Concentrations of both inhibitors vary from 0.5 up to 10 µM as indicated. Values were individually normalized for each concentration of inhibitors to the maximal current slope (recorded after FK stimulation) calculated between −100 and −60 mV and between +60 and +100 mV. Values are means (±SEM) of five to eight individual cells. *P < 0.05, Tukey's HSD test. The insets show the logarithmic dose–response curves corresponding to each inhibitor.
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fig02: Inhibition of forskolin-activated CFTR-like conductance by CFTRinh-172 and GlyH-101 inhibitors in CFTR-expressing cells. (A and B) Whole-cell current traces recorded in CFTR-expressing cells (kidney, DCT cells) under control condition and after forskolin exposure (FK, 1–10 µM). Once the Cl− conductance is fully developed (3–4 min), CFTRinh-172 or GlyH-101 were perfused at increasing concentrations (0.5, 1, 5, 10 µM). The membrane potential was held at −40 mV and currents were elicited by a train of 11 voltage steps (400 ms duration) between −100 and +100 mV in +20 mV increment. The zero current level is indicated by a dashed line. (C) Mean current/voltage relationships measured at 350 ms after the onset pulse corresponding to experiments performed (A and B) under control condition, after FK exposure and finally in the presence of CFTRinh-172 (10 µM) or GlyH-101 (10 µM). Values are means (±SEM) of six to eight individual cells. (D and E) Histograms illustrating the concentration-dependent inhibition of the CFTR conductance obtained with increasing concentrations of CFTRinh-172 (D) or GlyH-101 (E). Concentrations of both inhibitors vary from 0.5 up to 10 µM as indicated. Values were individually normalized for each concentration of inhibitors to the maximal current slope (recorded after FK stimulation) calculated between −100 and −60 mV and between +60 and +100 mV. Values are means (±SEM) of five to eight individual cells. *P < 0.05, Tukey's HSD test. The insets show the logarithmic dose–response curves corresponding to each inhibitor.

Mentions: To record only the CFTR-like Cl− conductance in the CFTR-expressing cell model, the endogenous CaCC-mediated current was impaired by the use of a high concentration of EGTA in the pipette solution and the extracellular bath solution was adjusted to 320 mosmol kg−1 H2O (addition of mannitol) to avoid activation of the VSORC conductance (Barriere et al., 2003). Under these experimental conditions, perfusion of forskolin (1–10 µM) rapidly induced (<4 min) the activation of a Cl− current exhibiting a linear current/voltage relationship (Figure 2A–C). Once the forskolin-activated current had reached a maximum, CFTRinh-172 or GlyH-101 were perfused at increasing concentrations (0.5, 1, 5, 10 µM, Figure 2A and B). CFTRinh-172 induced a reversible concentration-dependent inhibition of the CFTR-like current that was maximal at 5 µM (Figure 2A and D). Similarly, GlyH-101 induced a concentration-dependent inhibition of the CFTR-like current (Figure 2B and E). This inhibition was partly reversible on washing the cells (70% of recovery within 4 min) and showed a significant potential dependency (at 10 µM, the inhibition was more pronounced at positive potentials than at negative potentials, Figure 2B, C and E). Figure 2D and E summarises the inhibition for each concentration of the inhibitors. Values are expressed as a function of CFTR-maximal current slope (current slopes were calculated between −100 and −60 mV and between +60 and +100 mV). The concentration–response curve revealed an IC50 value below 1µM for CFTRinh-172 (0.74 and 0.56 µM for negative and positive potentials respectively) and varying between 3 µM at negative potentials and 0.87 µM at positive potentials for GlyH101. These results confirmed the ability of both drugs to inhibit efficiently the CFTR-mediated Cl− currents in mouse kidney cells.


Revisiting CFTR inhibition: a comparative study of CFTRinh -172 and GlyH-101 inhibitors.

Melis N, Tauc M, Cougnon M, Bendahhou S, Giuliano S, Rubera I, Duranton C - Br. J. Pharmacol. (2014)

Inhibition of forskolin-activated CFTR-like conductance by CFTRinh-172 and GlyH-101 inhibitors in CFTR-expressing cells. (A and B) Whole-cell current traces recorded in CFTR-expressing cells (kidney, DCT cells) under control condition and after forskolin exposure (FK, 1–10 µM). Once the Cl− conductance is fully developed (3–4 min), CFTRinh-172 or GlyH-101 were perfused at increasing concentrations (0.5, 1, 5, 10 µM). The membrane potential was held at −40 mV and currents were elicited by a train of 11 voltage steps (400 ms duration) between −100 and +100 mV in +20 mV increment. The zero current level is indicated by a dashed line. (C) Mean current/voltage relationships measured at 350 ms after the onset pulse corresponding to experiments performed (A and B) under control condition, after FK exposure and finally in the presence of CFTRinh-172 (10 µM) or GlyH-101 (10 µM). Values are means (±SEM) of six to eight individual cells. (D and E) Histograms illustrating the concentration-dependent inhibition of the CFTR conductance obtained with increasing concentrations of CFTRinh-172 (D) or GlyH-101 (E). Concentrations of both inhibitors vary from 0.5 up to 10 µM as indicated. Values were individually normalized for each concentration of inhibitors to the maximal current slope (recorded after FK stimulation) calculated between −100 and −60 mV and between +60 and +100 mV. Values are means (±SEM) of five to eight individual cells. *P < 0.05, Tukey's HSD test. The insets show the logarithmic dose–response curves corresponding to each inhibitor.
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fig02: Inhibition of forskolin-activated CFTR-like conductance by CFTRinh-172 and GlyH-101 inhibitors in CFTR-expressing cells. (A and B) Whole-cell current traces recorded in CFTR-expressing cells (kidney, DCT cells) under control condition and after forskolin exposure (FK, 1–10 µM). Once the Cl− conductance is fully developed (3–4 min), CFTRinh-172 or GlyH-101 were perfused at increasing concentrations (0.5, 1, 5, 10 µM). The membrane potential was held at −40 mV and currents were elicited by a train of 11 voltage steps (400 ms duration) between −100 and +100 mV in +20 mV increment. The zero current level is indicated by a dashed line. (C) Mean current/voltage relationships measured at 350 ms after the onset pulse corresponding to experiments performed (A and B) under control condition, after FK exposure and finally in the presence of CFTRinh-172 (10 µM) or GlyH-101 (10 µM). Values are means (±SEM) of six to eight individual cells. (D and E) Histograms illustrating the concentration-dependent inhibition of the CFTR conductance obtained with increasing concentrations of CFTRinh-172 (D) or GlyH-101 (E). Concentrations of both inhibitors vary from 0.5 up to 10 µM as indicated. Values were individually normalized for each concentration of inhibitors to the maximal current slope (recorded after FK stimulation) calculated between −100 and −60 mV and between +60 and +100 mV. Values are means (±SEM) of five to eight individual cells. *P < 0.05, Tukey's HSD test. The insets show the logarithmic dose–response curves corresponding to each inhibitor.
Mentions: To record only the CFTR-like Cl− conductance in the CFTR-expressing cell model, the endogenous CaCC-mediated current was impaired by the use of a high concentration of EGTA in the pipette solution and the extracellular bath solution was adjusted to 320 mosmol kg−1 H2O (addition of mannitol) to avoid activation of the VSORC conductance (Barriere et al., 2003). Under these experimental conditions, perfusion of forskolin (1–10 µM) rapidly induced (<4 min) the activation of a Cl− current exhibiting a linear current/voltage relationship (Figure 2A–C). Once the forskolin-activated current had reached a maximum, CFTRinh-172 or GlyH-101 were perfused at increasing concentrations (0.5, 1, 5, 10 µM, Figure 2A and B). CFTRinh-172 induced a reversible concentration-dependent inhibition of the CFTR-like current that was maximal at 5 µM (Figure 2A and D). Similarly, GlyH-101 induced a concentration-dependent inhibition of the CFTR-like current (Figure 2B and E). This inhibition was partly reversible on washing the cells (70% of recovery within 4 min) and showed a significant potential dependency (at 10 µM, the inhibition was more pronounced at positive potentials than at negative potentials, Figure 2B, C and E). Figure 2D and E summarises the inhibition for each concentration of the inhibitors. Values are expressed as a function of CFTR-maximal current slope (current slopes were calculated between −100 and −60 mV and between +60 and +100 mV). The concentration–response curve revealed an IC50 value below 1µM for CFTRinh-172 (0.74 and 0.56 µM for negative and positive potentials respectively) and varying between 3 µM at negative potentials and 0.87 µM at positive potentials for GlyH101. These results confirmed the ability of both drugs to inhibit efficiently the CFTR-mediated Cl− currents in mouse kidney cells.

Bottom Line: We also explored the effect of both inhibitors on cell viability using live/dead and cell proliferation assays in two different cell lines.The CFTRinh -172 did not affect the CaCC but did inhibit the VSORC, at concentrations higher than 5 µM.Our results provided insights into their use in mouse models.

View Article: PubMed Central - PubMed

Affiliation: University of Nice-Sophia Antipolis, LP2M CNRS-UMR7370, Faculté de médecine, Nice, France.

No MeSH data available.


Related in: MedlinePlus