Functional vacuolar ATPase (V-ATPase) proton pumps traffic to the enterocyte brush border membrane and require CFTR

Abstract

Vacuolar ATPases (V-ATPases) are highly conserved proton pumps that regulate organelle pH. Epithelial luminal pH is also regulated by cAMP-dependent traffic of specific subunits of the V-ATPase complex from endosomes into the apical membrane. In the intestine, cAMP-dependent traffic of cystic fibrosis transmembrane conductance regulator (CFTR) channels and the sodium hydrogen exchanger (NHE3) in the brush border regulate luminal pH. V-ATPase was found to colocalize with CFTR in intestinal CFTR high expresser (CHE) cells recently. Moreover, apical traffic of V-ATPase and CFTR in rat Brunner's glands was shown to be dependent on cAMP/PKA. These observations support a functional relationship between V-ATPase and CFTR in the intestine. The current study examined V-ATPase and CFTR distribution in intestines from wild-type, CFTR(-/-) mice and polarized intestinal CaCo-2BBe cells following cAMP stimulation and inhibition of CFTR/V-ATPase function. Coimmunoprecipitation studies examined V-ATPase interaction with CFTR. The pH-sensitive dye BCECF determined proton efflux and its dependence on V-ATPase/CFTR in intestinal cells. cAMP increased V-ATPase/CFTR colocalization in the apical domain of intestinal cells and redistributed the V-ATPase Voa1 and Voa2 trafficking subunits from the basolateral membrane to the brush border membrane. Voa1 and Voa2 subunits were localized to endosomes beneath the terminal web in untreated CFTR(-/-) intestine but redistributed to the subapical cytoplasm following cAMP treatment. Inhibition of CFTR or V-ATPase significantly decreased pHi in cells, confirming their functional interdependence. These data establish that V-ATPase traffics into the brush border membrane to regulate proton efflux and this activity is dependent on CFTR in the intestine.

Keywords: CFTR; V-ATPase; cAMP-regulated traffic; intestine.

Fig. 1.

Western blot analysis of endogenous vacuolar ATPase (V-ATPase) V_oa isoforms and V₁E subunit in polarized intestinal cells and rodent jejunum. Lysates were prepared from CaCo-2_BBe cells and rat and wild-type (WT) mouse jejunum (30 μg of protein). Proteins were separated by SDS-PAGE and detected by Western blot. Bands reveal patterns of endogenous V_oa1 or V_oa2 subunit isoforms and V₁E subunit in lysates. β-Actin loading controls are shown. Molecular mass standards (kDa) are indicated.

Fig. 2.

Western blot analysis of V-ATPase V_oa isoforms and V₁E subunit, cystic fibrosis transmembrane conductance regulator (CFTR), alkaline phosphatase (ALP), and sodium-coupled bicarbonate cotransporter 1 (NBCe1) in brush border membrane vesicle (BBMV) preparations from rat and WT mouse jejunum. Proteins from rat, WT mouse lysates, and BBMV (30 μg) were resolved by SDS-PAGE and detected with antibodies against V_oa1, V_oa2, V₁E, CFTR, ALP, galectin 4, and NBCe1. A: bands consistent with V_oa1, V_oa2, V₁E, CFTR, ALP, and galectin 4 are detected in BBMV preparations from rat and WT mouse jejunum. B: NBCe1 protein expression is detected in rat and WT mouse jejunum lysates but absent in rat and WT mouse BBMV preparations. Molecular mass standards (kDa) are indicated.

Fig. 3.

cAMP treatment increases the association of endogenous CFTR with V-ATPase V_oa isoforms and V₁E subunit in polarized CaCo-2_BBe cells. Coimmunoprecipitation of CFTR with V_oa1 or V_oa2 isoforms, V₁E subunit, and galectin 4 in CaCo-2_BBe cells treated with PBS, N⁶,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (DBcAMP; 1 mM), or PKA inhibitor (H-89; 10 μM) before DBcAMP (1 mM). CaCo-2_BBe cell lysates were immunoprecipitated with either anti-CFTR or control mouse IgG antibodies and bound to protein G beads. CaCo-2_BBe cell lysates (lysates, 30 μg), and equivalent loads of immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted to detect V_oa1, V_oa2, V₁E, galectin 4, CFTR, and β-actin. Western blot analysis of immunoprecipitates detects increased CFTR/V-ATPase association in DBcAMP-treated cells and decreased CFTR/V-ATPase association in DBcAMP-stimulated cells pretreated with H-89. Molecular mass standards (kDa) are indicated.

Fig. 4.

cAMP induces trafficking of endogenous V-ATPase V_oa isoforms or V₁E subunit and CFTR to the apical domain of CaCo-2_BBe cells. Confluent monolayers of CaCo-2_BBe cells were treated with PBS or DBcAMP (1 mM) for 30 min. Cells were fixed, immunolabeled with antibodies against CFTR and V-ATPase (V₁E, V_oa1, V_oa2, or V_oa3), and examined by confocal microscopy as described in materials and methods. Vertical XZ sections and en face views of immunolabeled cells following PBS and DBcAMP treatment reveal the distribution of V₁E (A, red), V_oa1 (B, red), V_oa2 (C, red), V_oa3 (D, red), or CFTR (green). Merged images show areas of colocalization (yellow). A higher magnification image taken from the inset shows areas of colocalization indicated in white. E: graphs show quantification of normalized apical CFTR, V₁E, V_oa1, V_oa2, or V_oa3 fluorescence intensity (FI) and percentage of CFTR colocalized with V₁E, V_oa1, V_oa2, or V_oa3 at the base of the microvilli in PBS- and DBcAMP-treated cells. White arrowhead in XZ images indicates section level for en face views and quantification. Data represent means +SE (n > 3). *P < 0.05. Scale bar = 10 or 100 μm.

Fig. 5.

H-89 inhibits cAMP-mediated trafficking of CFTR and V-ATPase V₁E subunit in polarized CaCo-2_BBe cells. Confluent monolayers of CaCo-2_BBe cells were treated for 30 min with PBS (A), DBcAMP (B; 1 mM), and PKA inhibitor (C; H-89, 10 μM). Cells were also pretreated with H-89 (D; 10 μM, 30 min) followed by DBcAMP (1 mM, 30 min). Cells were fixed, immunolabeled with antibodies against CFTR and V-ATPase (V₁E), and examined by confocal microscopy as described in materials and methods. A–D: vertical XZ sections and en face views of immunolabeled PBS and treated cells reveal the distribution of CFTR (green) or V₁E (red). Merged images show areas of colocalization (yellow). A higher magnification image taken from the inset shows areas of colocalization indicated in white. E: graphs show quantification of normalized apical CFTR and V₁E FI and percentage of CFTR colocalized with V₁E at the base of the microvilli. White arrowhead in XZ images indicates section level taken for enface views and quantification. Data represent means +SE (n > 3). *P < 0.05 for significant increase compared with PBS-, H-89-, and H-89 and DBcAMP-treated cells, and ^&P < 0.05 for significant increase compared with H-89-treated cells. Scale bar = 10 or 100 μm.

Fig. 6.

Concanamycin A (ConA) and CFTR _-inh172 inhibit cAMP-mediated trafficking of CFTR and V-ATPase V₁E subunit in polarized CaCo-2_BBe cells. Confluent monolayers were treated for 30 min with PBS (A), Streptolysin O (Slo; B; 15 nM), DBcAMP (C; 1 mM), Slo plus CFTR inhibitor, CF_-inh172 (D; 10 μM), or Slo plus the V-ATPase inhibitor ConA (F; 200 μM). Cells were also treated with CF_-inh172 (E; 10 μM, 30 min) or ConA (G; 200 μM) before treatment with DBcAMP (1 mM, 30 min). Cells were fixed and labeled with antibodies against CFTR and V₁E and examined by confocal microscope as described in the materials and methods. Vertical (XZ) merged images of CFTR (green) with V₁E (red) show areas of colocalization (yellow) on the apical membrane. En face views of immunolabled cells show the distribution of CFTR (green), V-ATPase V₁E (red), and merged images (yellow). A higher magnification image taken from the inset shows areas of colocalization indicated in white. H: graphs show quantification of normalized CFTR and V₁E FI and percentage of CFTR colocalized with V₁E at the base of the microvilli. White arrowhead in XZ images indicates section level taken for enface views and quantification. Data represent means +SE (n = 3). Normalized V₁E FI graph: *P < 0.05 for significant increase compared with PBS, Slo, Slo plus CF_-inh172, Slo plus ConA, and Slo plus ConA and DBcAMP-treated cells. ^&P < 0.05 for significant increase compared with H-89-treated cells. Normalized CFTR FI graph: *P < 0.05 for significant increase compared with PBS and all treated cells. ^#P < 0.05 for significant increase compared with PBS, Slo, and Slo plus CF_-inh172-treated cells. ^{^}P < 0.05 for significant increase compared with PBS, Slo, and Slo plus ConA-treated cells. Percentage of V₁E/CFTR colocalization graph: *P < 0.05 for significant increase compared with PBS and all treated cells. ⁺P < 0.05 for significant increase compared with Slo plus CF_-inh172-treated cells, and ^$P < 0.05 for significant increase compared with Slo plus ConA-treated cells. Scale bar = 10 or 100 μm.

Fig. 7.

V-ATPase proton efflux is CFTR dependent. CaCo-2_BBe cells were grown on coverslips, loaded with BCECF dye to measure intracellular pH over single cells, and the pH_i recovery rate was calculated from the slope after an acid load using a NH₄Cl prepulse as described in materials and methods. A: representative tracing from a single CaCo-2_BBe cell in culture. B: representative tracing from a CaCo-2_BBe cell that received the same NH₄Cl acidification prepulse, but the buffer was then exchanged for a Na and Cl⁻ free perfusate. C: representative tracing from a single CaCo-2_BBe cell that was acidified with NH₄Cl in the presence of ConA (200 μm), a selective inhibitor of the H-ATPase. D: summary graph shows the rate of proton movement/min across the membrane in each group. pH recovery requires Cl-dependent H-ATPase and CFTR. CaCo-2_BBe cells treated with Slo (15 nM) had no significant effect on the pH (2nd bar). The pH failed to recover in a Na and Cl⁻ free solution (3rd bar). CaCo-2_BBe cells treated with either Slo and the CFTR inhibitor CF_-inh172 (10 μM; 4th bar) or with Slo and ConA (200 μM; 5th bar) inhibited pH recovery. Treatment with Slo and DBcAMP (1 mM) increased proton efflux (6th bar). Each group is calculated from at least 5 different cells for each condition. Data are expressed as means ± SD in change in pH units/min. Statistical significance is shown on the graph.

Fig. 8.

cAMP-induced traffic of V-ATPase V_oa and V₁E subunits in WT and CFTR^−/− mouse jejunum. Intestinal loops from WT and CFTR^−/− mouse jejunum were treated with normal saline (NS) or DBcAMP (1 mM). Tissues were sectioned and double-labeled with antibodies against V_oa1, V_oa2, V_oa3, V₁E, and F-actin and examined by confocal microscopy as described in materials and methods. A–H: single and merged images show distribution of V_oa1, V_oa2 V_oa3, or V₁E in enterocytes from villus sections in NS and DBcAMP-treated WT (A, C, E, and G) and CFTR^−/− (B, D, F, and H) jejunum. Arrowheads indicate staining on the lateral membranes, and empty arrowheads indicate subapical staining in villus enterocytes. Hoechst stain labels nuclei blue. Scale bar = 10 um.

Fig. 9.

Western blot analysis of V-ATPase V_oa isoforms and V₁E subunit in WT and CFTR^−/− mouse jejunum. Tissue lysates (30 μg of protein) from WT and CFTR^−/− mouse jejunum were separated by SDS-PAGE. Proteins were detected using CFTR, V_oa1, V_oa2, V_oa3, or V₁E, and β-actin antibodies. Bands consistent with V_oa1 and V_oa2 isoforms, and V₁E isoform in WT and CFTR^−/− mouse jejunum are shown. Low levels of V_oa3 protein were detected. β-Actin loading controls are shown. Molecular mass standards (kDa) are indicated.

Fig. 10.

Schematic diagram summarizing effects of agonists and inhibitors on CFTR and V-ATPase function and regulated trafficking in polarized intestinal CaCo-2_BBe cells. A: schematic diagram illustrates proton movement across the apical membrane of cells. CF_-inh172 and/or ConA inhibit proton movement, whereas DBcAMP stimulates proton secretion across the membrane. B: schematic drawing illustrates cellular localization and apical trafficking patterns of CFTR and V-ATPase in intestinal cells. At steady state, CFTR is localized to the apical membrane and subapical endosomes, while V-ATPase is distributed throughout the cytoplasm and on the apical or basolateral domains. cAMP recruits both CFTR and V-ATPase to the apical membrane. The distribution of CFTR and V-ATPase in H-89 (PKA inhibitor)-, CF_-inh172-, and ConA-treated cells is similar to the localization patterns in steady-state conditions. In contrast, treatment with H-89, CF_-inh172, and ConA followed by DBcAMP induces trafficking of CFTR and V-ATPase to the apical membrane of the cells, indicating that CFTR is tightly linked to V-ATPase.