Cell Culture

16HBE14o- cells and Calu-3 cells (ATCC, VA, USA) were maintained in a minimal essential medium as described previously [9]. For simultaneous measurement of [Ca2+]i, or cAMP and short-circuit current (ISC), cells were seeded onto Transwell-COL membranes (0.4-µm pore size; Costar, Cambridge, MA, USA) with a culture area of 0.1 cm2 or 0.2 cm2. As previously described, we cultured primary HBEs (ScienCell Research Laboratories, CA, USA; Cat #3210, Lot #6457) from a single healthy donor at the ALI to produce pseudostratified mucociliary epithelia [8]. In brief, HBEs were grown in PneumaCult-Ex Plus Expansion Medium (#05040, STEMCELL, WA, USA). Subcultured HBEs were seeded on poly-L-lysine–coated Transwell permeable inserts. The cells were expanded before confluence using an expansion medium. Cells were air-exposed by removing the apical medium, and the basolateral medium was replaced with PneumaCult-ALI Maintenance Medium (#05001, STEMCELL, WA, USA) on day 0 (D0). A pseudostratified layer was obtained on ALI-D28. Transepithelial electrical resistance measurement was performed as described previously [8]. Similarly, both 16HBE14o- and Calu-3 cells were cultured under ALI conditions for 14 days post-confluence.

Exposing HBEs, 16HBE14o-, or Calu-3 Epithelia to e-Vapour or CS

ALI-cultured primary HBEs, 16HBE14o-, or Calu-3 epithelia were exposed to (i) e-vapour (30 or 70 watts [W]), (ii) CS (Camel cigarettes with filter), or (iii) air (control) using a Buxco Smoke Generation and Delivery System (Data Sciences International, MN, USA) similar to that described by Manevski et al. [10]. In the system, an electrically powered e-cigarette device (SMOK® X-Priv, Shenzhen IVPS Technology, SZ, PRC) generates e-vapour at various wattages. The system was also connected to an aerosol concentration measurement instrument (MicroDust Pro, Casella, Bedford, UK). Real-time monitoring of the total particulate matter (TPM) concentration generated by the vaping protocol of the smoking machine was performed before the e-vapour was delivered to the cultured cells inside the chamber. The concentration of the TPM generated at 30 and 70 W was relatively constant at 268.7 ± 49.9 and 833.3 ± 65.3 mg/m3 (n = 6), respectively. Three brands of e-liquid used were Reds Apple (Red; CA, USA), Saucy (SAUCY; CA, USA), and Ice (ICE; NRT, Japan). The e-liquids contained ~ 65% propylene glycol (PG), ~ 35% vegetable glycerine (VG), and food flavours with or without 20 mg/mL Nic. The SAUCY e-liquid had a Kiwi fruit, apple, and menthol flavour. Reds Apple was flavoured with berries, and Ice was flavoured with litchi. We used a high-puff volume and frequency protocol [11] comprising a 55-ml puff drawn over 3 s at 30-s intervals. Cells were exposed to e-vapour for 12 cycles prior to fluorescence and electrophysiological measurements. The puffing profile for CS exposure was the same as that used for e-vapour generation, ensuring that cells were exposed to the same amount of Nic. The total Nic content of two cigarettes consumed every 12 cycles was 1.6 mg. In the same 12 cycles, 0.08 mL of an e-liquid containing 20 mg/mL Nic was consumed, which is equivalent to 1.6 mg of Nic. The Nic content of e-cigarettes typically ranges from 3 to 36 mg/mL. The e-cigarettes of recent generations contain much more Nic (up to 60 mg/mL), usually in a salt form, so that Nic is delivered to the brain at a rate comparable to that of cigarette smoking [12]. Consequently, our e-liquid had a Nic content of 20 mg/mL. Based on the 65/35 ratio of PG to VG in e-liquids, we prepared a vehicle control of 65% PG and 35% VG (with/without 20 mg/mL Nic). Liquid Nic (≥ 99% (GC)) was added to the e-liquid to achieve the desired concentration. Following exposure, the epithelia were immediately transferred to a miniature Ussing chamber to measure intracellular calcium or cAMP levels and ISC.

Simultaneous Measurements of [Ca2+]i or cAMP and I SC

ATP- or UTP-induced Ca2+ signalling and anion secretion were measured simultaneously in polarised epithelia as described previously [9]. For ISC measurement, in brief, the monolayers were mounted in a miniature Ussing chamber and bathed in normal bicarbonate-buffered Krebs–Henseleit (KH) solution of the following composition (mM): NaCl, 117; NaHCO3, 25; KCl, 4.7; MgCl2, 1.2; KH2PO4, 1.2; CaCl2, 2.5; D-glucose, 11. The pH of the solution was 7.4 when the solution was bubbled with 5% CO2/95% O2. A basolateral-to-apical Cl− gradient favourable for apical Cl− exit was established across the monolayers by changing the apical KH solution to a low Cl− solution. In the low (10 mM) Cl− solution, NaCl, KCl, CaCl2, and MgCl2 were replaced isosmotically with Na-gluconate, K-gluconate, Ca-gluconate, and MgSO4, respectively. The potential difference was clamped to 0 mV, and ISC was simultaneously measured using a voltage clamp amplifier (VCC MC6; Physiologic Instruments, San Diego, CA, USA). A voltage pulse of 1 mV was applied periodically, and the resultant change in current was used in the calculation of the transepithelial resistance by Ohm’s law.

Imaging experiments used to measure intracellular cAMP and [Ca2+]i were conducted as described previously [13]. Real-time changes in the cAMP levels in living cells were monitored using Cyan Fluorescent Protein (CFP)-Epac-Yellow Fluorescent Protein (YFP), an Epac-based polypeptide fluorescence resonance energy transfer (FRET) reporter [14]. The monolayers were transfected with the Epac-based cAMP sensor for 2 days and then used for imaging experiments. FRET imaging experiments were performed with the constant perfusion of the KH solution at the apical and basolateral sides of the epithelia at 37 °C using an inverted microscope (Olympus IX70, Center Valley, PA, USA) with a 20 × /0.6 NA water immersion objective. The MetaFluor Imaging System with a FRET module (Molecular Devices, LLC, Sunnyvale, CA, USA) was used to control image acquisition. The cells were sequentially excited at 436 nm. The emission light was split by the Photometrics DV2 two-channel simultaneous imaging system (Photometrics, Tucson, AZ, USA), and emissions from CFP (470/30 nm filter) and YFP (FRET; 535/30 nm filter) were captured using a scientific CMOS camera (pco.edge 5.5; PCO AG, Kelheim, Germany). The acquired fluorescence images were background-corrected, and real-time changes in cAMP levels were represented by normalised CFP/FRET emission ratios. An increase in cAMP levels corresponded to an increase in the FRET ratio.

For [Ca2+]i measurement, the cells were loaded with 3 μM Fura-2-AM and excited at 340 and 380 nm; and Fura-2 emission (> 510 nm) was recorded, and the changes in [Ca2+]i were monitored by Fura-2 340/380 ratiometric imaging. A rise in [Ca2+]i correspondsed to an increase in the Fura-2 ratio. All signals were digitised, and data analysis was performed using the MetaFluor Imaging Software (v.7.5 with FRET module).

Western Blotting

16HBE14o-, Calu-3, and primary HBE monolayers were exposed to e-vapour for three consecutive days as described above and samples were collected 8 h after the third exposure to e-vapour for western blot analysis, which was performed as described previously [15]. For immunoblotting, the filter screen and filter paper were soaked in pre-cooled transfer buffer in advance. The gel and membrane (0.45 μm, PVDF) were cut according to the size of the target protein, soaked in methanol for 30 s for activation, and then soaked in pre-cooled transfer buffer. The transfer splint was installed according to the sandwich structure (filter screen–filter paper–gel–membrane–filter paper–filter screen) and correctly placed in the transfer tank according to the current direction. The transfer was conducted in an ice–water bath at a constant current of 300 mA for 2–4 h. After the transfer was complete, the PVDF membrane was taken out immediately, washed in a TBST washing solution for 1 min, and then blocked in a blocking solution at room temperature for 1 h. The primary antibodies were TMEM16A (#BP1-60076, 1:2000, NOVUS), NKCC1 (#8351, 1:2000, CST), KCNQ1 (#ab84819, 1:1000, Abcam), CFTR (#sc-376683, 1:1000, Santa Cruz, TX, USA), KCNN4 (#23271-1-AP, 1:2000, Proteintech, IL, USA), and β-ACTIN (#sc-8432, 1:3000, Santa Cruz). The secondary antibodies were goat anti-mouse (#31430, 1:10,000, Invitrogen) and goat anti-rabbit (#31460, 1:10,000, Invitrogen, MA, USA) HRP-conjugated secondary antibodies. Protein bands were visualised using the chemiluminescence substrate ECL (#1705061, Bio-Rad, CA, USA) and captured using a Bio-Rad ChemDoc Imaging System. Protein band intensities were quantified using the ImageJ programme and normalised to β-ACTIN.

Chemicals

The membrane-permeant acetoxymethylester (AM) form of Fura-2 was obtained from Molecular Probes (Eugene, OR, USA). ATP, UTP, Nic, and forskolin were obtained from Sigma-Aldrich (St. Louis, MO, USA). The laboratory reagents for general use were obtained from Sigma-Aldrich (St. Louis, MO, USA). All tissue culture reagents were obtained from Invitrogen.

Statistical Analysis

All data are expressed as means ± S.E.M., and values of n refer to the number of experiments in each group. Experimentally induced changes (∆) in Fura-2 ratios, FRET ratios, and ISC were measured at the peak of a response with subtraction of the values measured immediately prior to stimulation. Comparison of the means was performed using Student’s t-tests or one-way ANOVA (followed by post hoc tests) as appropriate using Graph Pad Prism 8 software (Prism, San Diego, CA, USA). A p-value of ≤ 0.05 was considered significant.