DETANO and nitrated lipids increase chloride secretion across lung airway cells

Abstract

We investigated the cellular mechanisms by which nitric oxide (NO) increases chloride (Cl-) secretion across lung epithelial cells in vitro and in vivo. Addition of (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETANONOate [DETANO];1-1,000 microM) into apical compartments of Ussing chambers containing Calu-3 cells increased short-circuit currents (I(sc)) from 5.2 +/- 0.8 to 15.0 +/- 2.1 microA/cm(2) (X +/- 1 SE; n = 7; P < 0.001). NO generated from two nitrated lipids (nitrolinoleic and nitrooleic acids; 1-10 microM) also increased I(sc) by about 100%. Similar effects were noted across basolaterally, but not apically, permeabilized Calu-3 cells. None of these NO donors increased I(sc) in Calu-3 cells pretreated with 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). Scavenging of NO either prevented or reversed the increase of I(sc). These data indicate that NO stimulation of soluble guanylyl cyclase was sufficient and necessary for the increase of I(sc) via stimulation of the apical cystic fibrosis transmembrane regulator (CFTR). Both Calu-3 and alveolar type II (ATII) cells contained CFTR, as demonstrated by in vitro phosphorylation of immunoprecipitated CFTR by protein kinase (PK) A. PKGII (but not PKGI) phosphorylated CFTR immuniprecipitated from Calu-3 cells. Corresponding values in ATII cells were below the threshold of detection. Furthermore, DETANO, 8-Br-cGMP, or 8-(4-chlorophenylthio)-cGMP (up to 2 mM each) did not increase Cl- secretion across amiloride-treated ATII cells in vitro. Measurements of nasal potential differences in anesthetized mice showed that perfusion of the nares with DETANO activated glybenclamide-sensitive Cl- secretion. These findings suggest that small concentrations of NO donors may prove beneficial in stimulating Cl- secretion across airway cells without promoting alveolar edema.

Figure 1.

Nitrolinoleic acid (LNO₂) and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETANO) increase short-circuit current (I_sc) in Calu-3 cells. Confluent monolayers of Calu-3 cells were mounted in Ussing chambers. LNO₂ (0.01–10 μM; left panels) or DETANO (1–1,000 μM; right panels) were added into apical compartments of the Ussing chambers. In paired experiments, equivalent amounts of the parent compound (linoleic acid [LA]) or vehicle (NaOH) were added in similar fashion. (A and C) Typical recordings of I_sc in individual experiments. Mean values ± 1 SE are shown in Table 1. (B and D) Percent of maximum I_sc versus concentration of LNO₂ or DETANO; percent of maximum I_sc = ((Ix − I₀)/(I_max − I₀)) × 100, where I₀ is the I_sc obtained in the absence of LNO₂ or DETANO, I_x is the steady-state current at a given concentration (μM) of LNO₂ or DETANO, and I_max is the steady-state value of I_sc after addition of either 10 μM LNO₂ or 1,000 μM DETANO. Data were fitted by spline curves using Slide Write for Windows 6. Values for 50% stimulation (∼0.06 μM for LNO₂ and 12 μM for DETANO) were calculated by extrapolation from these figures.

Figure 2.

1H-(1, 2, 4)-oxadiazolo (4, 3-a) guinoxalin-1-one (ODQ) prevents the effect of nitric oxide (NO)–induced I_sc in the Calu-3 cells. Addition of either 10 μM LNO₂ or 50 μM DETANO increased I_sc, which was reversed by oxymyoglobin (oxyMb; 400 μM). Pretreatment of Calu-3 cells with ODQ (10 μM) twice a day for 3 days prevented the increased of I_sc by either LNO₂ or DETANO (A and B). However, addition of CPT-cAMP (0.05 mM) into the apical compartment after ODQ increased I_sc, indicating that ODQ did not cause nonspecific cellular damage. Results of typical experiments are shown; for mean values ± 1 SE see Table 3.

Figure 3.

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors reversed the NO increase of I_sc in Calu-3 cells. Glybenclamide (0.3 mM) or CFTR inhibitor-172 (CFTR_inh172; 1–100 μM) added into the apical compartments of Calu-3 cells rapidly reversed the increase of I_sc by either LNO₂ (10 μM; left panel) or DETANO (50 μM; right panel). Results of typical experiments are shown; for mean values ± 1 SE see Table 4.

Figure 4.

LNO₂ and DETANO increase I_sc across basolaterally permeabilized Calu-3 monolayers. Confluent monolayers of Calu-3 cells were mounted in Ussing chamber containing nonsymmetrical chloride (Cl⁻) solutions (apical, 5 mM; basolateral, 125 mM). Amphotericin B (10 μM) and ouabain (1 mM) were added into the basolateral compartment, followed 10 to 20 minutes later by LNO₂ (10 μM; A–C) or DETANO (50 μM; D). Addition of oxyMb (0.4 mM; A, C, and D) or glybenclamide (0.3 mM; B) into the apical compartments rapidly decreased I_sc. Pretreatment of Calu-3 cells with oxyMb (0.4 mM; A) or glybenclamide (0.3 mM; B) and ODQ (10 μM) twice a day for 3 days prevented the increased of I_sc by either LNO₂ or DETANO (C and D). Results of typical experiments are shown. Mean values ± 1 SE are shown in Tables 5 and 6.

Figure 5.

Measurements of NO release by DETANO. DETANO (50 μM) was added into a reaction chamber containing a confluent monolayer of Calu-3 cells. OxyMb was added at times indicated by arrows. (A) Time course of oxyMb inhibition of NO levels (measured by the ISO-NO meter). (B) oxyMb inhibition of I_sc after addition of DETANO. Results are shown of typical experiments that were repeated five different times.

Figure 6.

Measurements of NO release by LNO₂. (A) Difference spectra for the oxidation of oxyMb (25 μM) to metmyoglobin (metMb) by LNO₂ (10 μM) taken at 10-minute intervals, showing the characteristic decreases in the 582- and 544-nm maxima (arrows). The absorbance at the isosbestic wavelength for the oxyMb-metMb conversion (525 nm) was set to zero. (B) Time course of NO generation, as determined by the decrease in absorbance at 582 nm, is shown. Data represent means ± SE for three different experiments. (C) Time course of I_sc increase after addition of LNO₂ (1 μM) into the apical chamber of an Ussing chamber containing Calu-3 cells. Addition of oxyMb (1–100 μM) rapidly reversed the increase of I_sc; in a different set of experiments, addition of oxyMb (100 μM) into the apical chambers prevented the increase of I_sc by 10 μM LNO₂ (data not shown). (D) ΔI_sc, calculated by subtracting the plateau value of I_sc after addition of the indicated amount of oxyMb (x axis) from the Isc after addition of 1 μM LNO₂ (see C). Negative values indicate a decrease of I_sc. Mean values ± 1 SE; n = 3; *P < 0.05 compared to the absence of oxyMb (using one- and two-tailed t tests, respectively).

Figure 7.

Measurements of NO release by nitrooleate (ONO₂). (A) Difference spectra for the oxidation of oxyMb (25 μM) to metMb by ONO₂ (10 μM) taken at 10-minute intervals, showing the characteristic decreases in the 582- and 544-nm maxima (arrows). The absorbance at the isosbestic wavelength for the MbO₂-metMb conversion (525 nm) was set to zero. (B) Time course of NO generation, as determined by the decrease in absorbance at 582 nm. Data represent means ± SE for three different experiments. (C) Black line, time course of I_sc increase after addition of ONO₂ (1 μM) into the apical chamber of an Ussing chamber containing Calu-3 cells; grey line, same as above, except that oxyMb (100 μM) was added into the apical compartments before ONO₂. (D) ΔI_sc, calculated by subtracting the plateau value of I_sc after addition of the indicated amount of oxyMb (x axis) from the I_sc after addition of 1 μM ONO₂ (see C). Negative values indicate a decrease of I_sc. Mean values ± 1 SE; n = 3; *P < 0.05 compared to the corresponding value in the absence of oxyMb (using one- and two-tailed t tests, respectively).

Figure 8.

Immunocytochemical detection of CFTR in Calu-3 (left panels) and rat alveolar type II (ATII) cells (right panels). Calu-3 (A–D) and rat ATII (E–H) cells were immunostained with either a mouse monoclonal anti-CFTR antibody (B, D, F, and H) or with an equivalent amount of nonimmune mouse IgG (A, C, E, and G) followed by a secondary antibody, goat anti-mouse IgG Alexa 488 (green fluorescence). Nuclei were stained with Hoechst 33258 dye. In some cases (A, B, E, and F), the filters were folded to visualize the apical membranes. Characteristic figures were chosen from either 4 (folded) or 10 (nonfolded) different records.

Figure 9.

In vitro phosphorylation of immunoprecipitated CFTR from Calu-3 and ATII cells by protein kinase (PK) A. Calu-3 and ATII cells were grown on permeable supports. When confluent, CFTR was immunoprecipitated with anti-(M3A7) monoclonal antibody and in vitro–phoshorylated using γ³²P ATP and PKA, separated by SDS-PAGE, and detected with autoradiography as described in the text. To enhance detection, we used about twice as much protein from ATII than from Calu-3 cells for immunoprecipitation. Results presented are from a typical experiment, repeated twice with similar results.

Figure 10.

Phosporylation of CFTR by PKGII. (A) Cellular proteins from either Calu-3 or ATII cells were separated with SDS-PAGE, transferred to polyvinylidene fluoride membranes, and immunostained with PKGII antibodies (lanes 1–3), PKGII antibodies along in the presence of the immunizing peptide (lane 4) or PKGI antibodies (lanes 6–7); positive control (rat brain extract; lane 5). Results are shown of a typical experiment that was repeated three times. (B) Calu-3 cells expressing wild-type CFTR were grown on plastic dishes. Cells were lysed in RIPA buffer and CFTR was immunoprecipitated from equal amounts of cells using 24-1 and protein A Agarose. CFTR was in vitro phosphorylated with PKA (lane 1), PKGI (lane 3), PKGII (lanes 4 and 6) and PKGI plus PKGII (lane 5) according to the manufacturer's recommendations. Results are those of a typical experiment that was repeated three times. Grey arrow above the 75-kD MW marker shows the location of PKGII; white arrow (right) shows the location of PKGI.

Figure 11.

DETANO increases Cl⁻ secretion across nasal airways in vivo. Recordings of nasal potential differences (NPDs) in Balb/c mice perfused with 6 mM Cl⁻ solution containing 100 μM amiloride to block Na⁺ absorption and enhance Cl⁻ secretion. (A) Addition of 0.1 mM DETANO (or 0.5 mM; data not shown) resulted in significant hyperpolarization (indicative of enhanced Cl⁻ secretion), which was blocked partially by glybenclamide (0.3 mM). (B) Addition of equal concentrations of oxyMb and DETANO (0.1 mM) into the perfusate had no effect on NPD. (C) Mean values of NPD changes (±1 SE; n = 3); *P < 0.05 compared to vehicle; ^#P < 0.05 compared to DETANO. Dotted lines indicate baseline levels before addition of DETANO

Figure 12.

cAMP but not cGMP increased Cl⁻ I_sc across rat ATII cells. Confluent monolayers of rat ATII cells were mounted in Ussing chambers as described in Materials and Methods. (A) Addition of amiloride (100 μM; an inhibitor of epithelial Na⁺ channels) into the apical compartment rapidly decreased I_sc, indicating that the majority of I_sc is due to the movement of Na⁺ ions. cAMP (100 μM CPT-cAMP, 10 μM forskolin, and 100 μM IBMX), added into the apical compartments, increased I_sc, which was either reversed (reversed) or prevented (grey line) by the CFTR_inh172 (20 μM). (A–D) DETANO (1–1,000 μM; B), Bay 41-2272 (1–100 μM; C), Br-cGMP (1–2,000 μM; D), or CPT-cGMP (1–2,000 μM; data not shown) did not increase I_sc after addition of amiloride. Addition of CFTR_inh172 (1–200 μM; grey line in D) into the apical compartment decreased I_sc, indicating that the majority of I_sc is also due to the movement of Cl⁻ ions. Results are those of typical experiments that were repeated at least three times each. Mean values ± 1 SE are shown in Table 7.