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. 2018 Jul 13;7:e37963. doi: 10.7554/eLife.37963

Figure 7. PKA-dependent channel activity of CFTR is enhanced by NO dependent activation of PKG in colonic epithelium.

(a) Model depicting the hypothesis that SLC6A14-mediated arginine uptake across the epithelium would lead to an increase in intracellular NO levels, which would then potentiate CFTR channel function via PKG activation. (b) Line graph represents CFTR function measured using the FLIPR® membrane potential assay in Caco-2 colonic intestinal epithelial cells. Cells were pre-treated with PKG inhibitor KT5823 (1 µM) or DMSO control. After reading baseline intracellular NO levels were increased using NO donor GSNO (10 µM) or vehicle control, followed by cAMP activation of CFTR using FSK (10 µM) or DMSO vehicle. (c) Bar graph represents response to FSK from baseline (ΔF/F0), in Caco-2 cells pre-treated with PKG inhibitors Rp-8-pCPT-cGMPS (RP8), KT5823 or vehicle DMSO, followed by addition of NO donor GSNO as shown in Figure 7b. The effect on FSK response by another NO donor SNP (50 µM) and direct increase of intracellular cGMP (10 µM) was also studied in this system. Mean ± SEM are plotted. One-way ANOVA with Tukey’s multiple comparison test was performed (*p=0.006, **p=0.0001, # p=0.004, # # p=0.002, n = 3 biological replicates). (d) Bar graph represents CFTRinh-172 (10 µM) response, elicited at the end of the experiment after full CFTR activation. Mean ± SEM are plotted. Unpaired t-test was performed (*p=0.007, n = 3 biological replicates). (e) Line graph represents CFTR function measured using the FLIPR® membrane potential assay in Caco-2 epithelial cells transduced with SLC6A14-GFP or control GFP alone. Cells were pre-treated with PKG inhibitors Rp-8-pCPT-cGMPS (10 µM), KT5823 (1 µM), or iNOS inhibitor 1400W (50 µM) or DMSO control. After reading for baseline, SLC6A14 was stimulated using L-arginine (1 mM) or vehicle control, followed by cAMP activation of CFTR using FSK (10 µM) or DMSO vehicle. This was followed by addition of CFTRinh-172 (10 µM). (f) Bar graph represents CFTRinh-172 (10 µM) response, elicited at the end of the experiment after full CFTR activation, in Caco-2 cells over-expressing SLC6A14-GFP or control GFP. Mean ±SEM are plotted. One-way ANOVA with Tukey’s multiple comparison test was performed (****p<0.0001, **p=0.003, ***p=0.0001, n = 3 biological replicates).

Figure 7.

Figure 7—figure supplement 1. SLC6A14-GFP over-expression in Caco2 colonic epithelial cells.

Figure 7—figure supplement 1.

(a) SLC6A14 expression in Caco2 cells transduced with SLC6A14-GFP or GFP alone, was measured. SLC6A14 mRNA expression normalized to housekeeping gene GUSB was measured using PCR for 40 cycles. Amplification of SLC6A14 cDNA segment was observed (175 bp) in Caco2 cells over-expressing SLC6A14-GFP, but not in the control over-expressing GFP alone. Housekeeping gene GUSB (197 bp) was present in both the samples. (b) Bars represent relative mRNA expression of SLC6A14 normalized to housekeeping gene GUSB (mean ± SEM, unpaired t-test, *p=0.001, n = 3 technical replicates). (c) Caco2 cells overexpressing SLC6A14-GFP or GFP alone were lysed and subjected to western blotting. The blot was probed with anti-GFP antibody. (d) Caco2 cells overexpressing SLC6A14-GFP or GFP alone were lysed and subjected to western blotting. The blot was probed with anti-CFTR (596) and anti-calnexin antibodies.
Figure 7—figure supplement 2. SLC6A14-GFP functions as an electrogenic amino-acid (arginine) transporter.

Figure 7—figure supplement 2.

(a) Caco-2 cells transduced with SLC6A14-GFP or control (GFP only) were assessed for functional expression of SLC6A14-GFP as an electrogenic amino acid transporter. Line graph represents FLIPR® based membrane potential change in these cells at baseline and upon acute addition of L-arginine (1 mM). (b) Bar graph represents response to L-arginine measured as maximum change in fluorescence (ΔF) relative to baseline (F0), in Caco-2 cells over-expressing SLC6A14-GFP or control (GFP only). (c) CFBE cells transduced with SLC6A14-GFP localizes to the apical surface, to the same plane as ZO-1 (red). (d) 3H-arginine uptake was mediated by CF bronchial epithelial cells (CFBE’s) transduced with SLC6A14-GFP. Bar graph represents mean ± SD of arginine uptake by the epithelium (mol/minute/mg ofprotein). Alpha-methyl-tryptophan (α-MT 1 mM), an inhibitor of SLC6A14, significantly inhibited uptake. Unpaired t-test (*p<0.0001, n = 3 biological replicates defined as different cell passages).