Hydrogen peroxide stimulation of CFTR reveals an Epac-mediated, soluble AC-dependent cAMP amplification pathway common to GPCR signalling

Abstract

Background and purpose: H2 O2 is widely understood to regulate intracellular signalling. In airway epithelia, H2 O2 stimulates anion secretion primarily by activating an autocrine PGE2 signalling pathway via EP4 and EP1 receptors to initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion. This study investigated signalling downstream of the receptors activated by H2 O2 .

Experimental approach: Anion secretion by differentiated bronchial epithelial cells was measured in Ussing chambers during stimulation with H2 O2 , an EP4 receptor agonist or β2 -adrenoceptor agonist in the presence and absence of inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca(2+) ]I ) changes were followed by microscopy using fura-2-loaded cells and PKA activation followed by FRET microscopy.

Key results: Transmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were both necessary for H2 O2 and EP4 receptor-mediated CFTR activation in bronchial epithelia. H2 O2 and EP4 receptor agonist stimulated tmAC to increase exchange protein activated by cAMP (Epac) activity that drives PLC activation to raise [Ca(2+) ]i via Ca(2+) store release (and not entry). Increased [Ca(2+) ]i led to sAC activation and further increases in CFTR activity. Stimulation of sAC did not depend on changes in [HCO3 (-) ]. Ca(2+) -activated apical KCa 1.1 channels and cAMP-activated basolateral KV 7.1 channels contributed to H2 O2 -stimulated anion currents. A similar Epac-mediated pathway was seen following β2 -adrenoceptor or forskolin stimulation.

Conclusions and implications: H2 O2 initiated a complex signalling cascade that used direct stimulation of tmACs by Gαs followed by Epac-mediated Ca(2+) crosstalk to activate sAC. The Epac-mediated Ca(2+) signal constituted a positive feedback loop that amplified CFTR anion secretion following stimulation of tmAC by a variety of stimuli.

Figure 1

H₂O₂ stimulates both tmAC and sAC. Fully differentiated NHBE cells in ALI culture were mounted in Ussing chambers and stimulated with either H₂O₂ (1 mM) or forskolin (10 μM) in the presence of various concentrations of the tmAC inhibitor MDL-12,330A (A, n = 3–6 lung donors at each concentration) or various concentrations of the sAC inhibitor KH7 (B, 3–6 lung donors at each concentration). (C) NHBE cells were infected with shRNA expressing lentiviruses targeted to either exon 2 or exon 15 of sAC or with non-targeted lentiviruses. After differentiation, cultures were mounted in Ussing chambers and stimulated with H₂O₂ (1 mM). Compared with non-target controls and exon 2-targeted cultures, the response of exon 15 targeted cultures was significantly reduced (n = 5 cultures from two lung donors, *P < 0.05).

Figure 2

H₂O₂-mediated [Ca²⁺]_i increases stimulate anion secretion. (A) Fully differentiated NHBE cells in ALI culture were loaded with fura-2AM and mounted in a perfusion chamber on a Nikon E600fn microscope (Nikon Inc., Melville, NY, USA). H₂O₂ (400 μM) led to an increase in [Ca²⁺]_i. Subsequent perfusion with ATP (10 μM) led to the expected robust response (ratio plotted is the mean of 16 cells from 1 donor ± SEM, representative of experiments with three individual lungs). For comparison of ATP-stimulated I_SC changes, see Supporting Information Fig. S3. (B) Representative I_SC traces of fully differentiated NHBE cells in ALI culture loaded with 25 μM BAPTA-AM (solid trace) before they were mounted in Ussing chambers and stimulated with H₂O₂ (1 mM) compared with control (unloaded cells; dashed trace). (C) BAPTA-loaded cultures had a reduced anion secretion response to 1 mM H₂O₂ (mean ± SEM, n = 6 lung donors, two to three cultures per donor, *P < 0.05). (D) NHBE ALI cultures in Ussing chambers were pretreated with different concentrations of the PLC inhibitor U73122 and then stimulated with H₂O₂ (1 mM) in the presence of inhibitor. U73122 led to a concentration-dependent decrease in anion secretion with an apparent IC₅₀ = 10 μM (n = 3–4 lung donors at each concentration). (E) Comparison of U73122 (25 μM) with the less active isomer U73343 (25 μM) showed specificity. (F) NHBE ALI cultures were mounted in Ussing chambers and stimulated with H₂O₂ (1 or 0.4 mM) in the presence or absence of extracellular Ca²⁺, in the presence of Sr²⁺ instead of Ca²⁺ or in the presence of 2-APB (200 μM). Neither removal of Ca²⁺ nor substitution of Ca²⁺ with Sr²⁺ significantly reduced anion secretion (n = 3 lung donors), while addition of the IP₃ receptor antagonist 2-APB significantly reduced anion secretion (n = 5 lung donors, *P < 0.05).

Figure 3

H₂O₂ stimulation of sAC is not HCO₃⁻ dependent. (A) Representative I_SC traces of fully differentiated NHBE cells in an ALI culture mounted in an Ussing chamber containing KH without HCO₃⁻, buffered with 10 mM HEPES pH 7.4 and stimulated with 1 mM H₂O₂ in the absence (dashed trace) or presence of KH7 (solid trace, 25 μM). (B) Removal of HCO₃⁻ did not reduce anion secretion in response to H₂O₂ (n = 5 lungs), and the H₂O₂ response was sensitive to KH7 in the absence of bicarbonate (n = 3 lungs, *P < 0.05).

Figure 4

EP4 stimulation of CFTR activates sAC through Epac and increased [Ca²⁺]_i. (A) NHBE ALI cultures in Ussing chambers were stimulated with Cay10598 (50 nM) in the presence or absence of H89 (10 μM), CFTR_inh172 (10 μM) or 4,4′-dinitro-stilbene-2,2′-disulphonic acid (DNDS; 100 μM) (n = 4 lung donors for each inhibitor, *P < 0.05). Both the kinase inhibitor H89 and the CFTR inhibitor blocked EP₄ receptor-mediated anion secretion, while DNDS has no effect, consistent with CFTR activation. (B) I_SC traces of NHBE ALI cultures mounted in Ussing chambers and stimulated with Cay10598 (50 nM) in the presence or absence of KH7 (25 μM) or MDL12,330A (25 μM). (C) Anion secretion was significantly reduced by each inhibitor (n = 5 lung donors, *P < 0.05). (D) NHBE ALI cultures were stimulated with Cay10598 in the presence of different concentrations of KH7 (n = 3–6 lung donors at each concentration, apparent IC₅₀ = 8 μM). (E) NHBE ALI cultures were loaded with fura2-AM, mounted in a perfusion chamber and imaged by epifluorescence microscopy. Addition of Cay10598 (100 nM) to the perfusate increased [Ca²⁺]_i that returned towards baseline after removal of the agonist (representative trace is the mean fura-2 ratio recorded from regions of interest in 16 cells from a single lung donor ± SEM, and is representative of experiments with three individual donors). (F) The Epac inhibitor ESI-09 blocked H₂O₂-induced changes in I_SC with an apparent IC₅₀ = 0.8 μM (n = 1–5 lung donors at each concentration), and ESI-09 (10 μM) attenuated Cay10598-induced anion secretion (G; n = 4 lung donors, *P < 0.05 compared with control no ESI-09). (H) Pretreatment with ESI-09 (10 μM) also attenuated changes in Cay10598-induced fura-2 fluorescence (representative trace of the mean fura-2 ratio recorded from regions of interest in 19 cells from a single donor ± SEM, and is representative of experiments with three individual donors). (I) Cay10598-induced fura-2 fluorescence changes were significantly altered by ESI-09 (10 μM; n = 4 lung donors, P < 0.05).

Figure 5

Epac inhibition partially blocks forskolin stimulation of CFTR. (A) NHBE ALI cultures in Ussing chambers were stimulated with Cay10598 (100 nM, squares), albuterol (10 μM, circles) or forskolin (10 μM, triangles) in the presence of KH7. Forskolin trace is from Figure 1A. Values plotted are the mean ± SEM, n = 3–6 lung donors at each point. (B) NHBE ALI cultures that were transduced with lentiviruses encoding fluorescent PKA subunits, mounted in a perfusion chamber in the presence or absence of ESI-09 (10 μM) and imaged by epifluorescence microscopy during forskolin (10 μM) stimulation. Shown are example traces from a single control cell (black trace) and a single-cell pretreated with ESI-09 (grey trace). (C) Maximum forskolin-induced changes in the FRET ratio are shown with and without ESI-09 (mean ± SEM, n = 17, eight to nine cells from each of two donors). (D, E) NHBE ALI cultures in Ussing chambers were stimulated with 8-pCPT-2′-O-Me-cAMP (20 μM) and the increased I_SC was blocked by addition of KH7 (25 μM, D), by BAPTA loading (D, grey trace) and by CFTRinh172 (5 μM, E). (F) NHBE ALI cultures in Ussing chambers stimulated with 8-pCPT-2′-O-Me-cAMP and treated with KH7 or were stimulated with 8-pCPT-2′-O-Me-cAMP with or without loading with BAPTA-AM (n = 3 lung donors).

Figure 6

H₂O₂-mediated stimulation of CFTR via sAC occurs in basolaterally permeabilized cells. (A) Representative I_SC traces of NHBE cells in ALI culture mounted in Ussing chambers and stimulated with H₂O₂ (1 mM) with (solid trace) or without Ba²⁺ (dashed trace, 5 mM). (B) NHBE ALI cultures mounted in Ussing chambers and stimulated with H₂O₂ in the presence or absence of basolateral Ba²⁺ (5 mM), clofilium (100 μM), clotrimazole (30 μM) or apical paxilline (4 μM). Compared with controls basolateral Ba²⁺ and clofilium and apical paxilline blocked H₂O₂ responses while clotrimazole had no effect (n = 5–8 lung donors, *P < 0.05). (C) NHBE cells in ALI culture were mounted in buffer containing 100 μM nystatin to permeabilize the basolateral membrane and with or without MDL-12,330A (75 μM) or KH7 (20 μM). After stabilization of baseline I_SC, H₂O₂ was added to the apical compartment. KH7 inhibited H₂O₂ stimulation while MDL-12,330A inhibition was reduced by basolateral membrane permeabilization (n = 3 lung donors, *P < 0.05 compared with controls).

Figure 7

Proposed model for H₂O₂ stimulation of CFTR. In differentiated NHBE cells, H₂O₂ activates the GPCRs EP1 and EP4. EP4, that appears responsible for the majority of the H₂O₂ response (Conner et al., 2013), and albuterol both activate Gαs to stimulate tmAC. Increased cAMP activates PKA to stimulate CFTR-mediated Cl⁻ secretion and basolateral K_v7.1 K⁺ secretion. Also, cAMP activation of Epac initiates a Ca²⁺ signal presumably through PLC stimulation. This Epac-dependent Ca²⁺ signal stimulates both tmAC and sAC to amplify the cAMP/PKA pathway.