5-nitro-2-(3-phenylpropylamino)benzoic acid

Macrophages (~1× 10⁵ cells), attached to glass coverslips precoated with Cell Tak (BD scientific laboratories, San Jose, CA), were incubated for 30 min at 37°C with N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide (MQAE-30mM) (17 (link)). MQAE (Invitrogen, Carlsbad, CA) is a Cl⁻ sensitive fluorescent indicator dye that measures increases in Cl⁻ concentration via a quenching mechanism. Reductions in cell Cl⁻ give increases in fluorescent intensity indicative of decreased cytosolic Cl⁻ concentration (18 (link)). Dye loading and subsequent experimentation were performed in a custom perfusion chamber mounted on an Olympus IX-71 inverted microscope (19 (link)). MQAE was excited at 354 ± 10 nm and emitted fluorescent light was measured at 460 ± 10 nm every 5s using a charge coupled device camera attached to a digital imaging system (20 (link), 21 (link)). Typically, 10–20 macrophages were monitored simultaneously for each experiment. The rate of change in MQAE fluorescence (Δarbitrary fluorescent units (AFU)/Δtime(s)) was used to calculate Cl⁻ efflux.
Initially macrophages were perfused at 3–4 ml/min with Cl⁻-containing solution (135 mM NaCl, 5mM KCl, 1 mM CaCl₂, 1.2 mM MgSO₄, 2mM NaH₂PO₄, 2mM HEPES, 10mM glucose or previously described (20 (link))) to allow for removal of extraneous dye. Following the initial perfusion with Cl⁻-containing buffer, the perfusate was changed to a Cl⁻-free solution (135 mM NaCyclamate, 3mM KGluconate, 0.5 mM CaCyclamate, 1.2 mM MgSO₄, 2mM KH₂PO₄, 2mM HEPES, 10mM Glucose or previously described (20 (link))) in which Cl⁻ was substituted with cyclamate. In a subset of experiments the loading of MQAE was assessed by exposing cells to a final perfusion solution containing potassium thiocyanate (KSCN) (150 mM KSCN, 0.5 mM CaCyclamate, 1.2 mM MgSO₄, 2mM KH₂PO₄, 2mM HEPES, 10mM Glucose) with Nigericin (10 µM) to measure the minimum specific fluorescence of the cells (20 (link)). The control Cl⁻-free solution, contained 0.5mM Ca²⁺, which is within the normal range for extracellular Ca²⁺ concentrations (22 (link)–25 (link)). The high Ca²⁺/Cl⁻-free solution had a Ca²⁺ concentration of 2mM, which can be found in tracheobronchial secretions (26 (link), 27 (link)). Macrophages were assessed in a low Ca²⁺ (0.1mM)/Cl⁻-free solution for comparison. To ensure that the extracellular Ca²⁺ concentrations did not affect cell viability, assays were performed with Trypan blue in each experimental solution demonstrating ≥90% viability. Solutions were adjusted to a final pH of 7.4 at 37°C and an osmolarity of 300mOsmol.
To confirm the presence of Cl⁻ movement, macrophages were assessed in the presence of 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB-100 µM), a broad inhibitor of Cl⁻ channels (28 (link)), as cells transitioned from Cl⁻-containing to Cl⁻-free solutions. The contribution of CFTR to total Cl⁻ flux was evaluated in the presence of the CFTR-specific inhibitor, cftr_inh-172 (20µM). Macrophages were treated for 2 min with cftr_inh-172 in the Cl⁻-containing solution, prior to assessing Cl⁻ efflux in the control Cl⁻-free solution with cftr_inh-172 still present. Rates of Cl⁻ efflux following treatment with either inhibitor were compared with rates of Cl⁻ efflux observed in the absence of the inhibitors. Vehicles alone (ethanol or DMSO) had no effect on efflux.
The effect of increasing intracellular Ca²⁺ concentrations on Cl^– efflux were assessed indirectly following treatment with either carbachol or thapsigargin. Macrophages were treated with carbachol (100 µM) for 30 min, while loading with MQAE (29 (link)). Alternatively, macrophages were assessed following treatment with thapsigargin (1 µM) for 2 min in Cl⁻-containing solution prior to assessment in Cl⁻-free solution. Following treatment with either agent, Cl^– efflux was assessed in either low Ca²⁺/Cl⁻-free solution with addition of EGTA (1mM) or in the control solution. Chemicals were purchased from Sigma Corporation unless specified.