Epithelial organic cation transporters ensure pH-dependent drug absorption in the airway

Abstract

Most inhaled beta(2)-adrenergic agonist and anticholinergic bronchodilators have low lipid solubility because of their transient or permanent positive net charge at physiologic pH. Airway absorption of these cationic drugs is incompletely understood. We examined carrier-mediated mechanisms of cationic drug uptake by human airway epithelia. Airway tissues and epithelial cells, obtained from lung donors without preexisting lung disease, were evaluated for organic cation transporter expression by quantitative RT-PCR and immunofluorescence. For in vitro functional studies on primary airway epithelial cells, uptake of the cationic fluorophore 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP+) was characterized. Quantitative RT-PCR analysis demonstrated high mRNA levels for two polyspecific organic cation/carnitine transporters, OCTN1 and OCTN2, in human airway epithelia. Immunofluorescence of human airway sections confirmed OCTN1/2 protein expression, with a predominant localization to the apical portion of epithelial cells. Primary airway epithelial cells showed a carrier-mediated, temperature-sensitive and saturable uptake of ASP(+). Seventy-five to eighty percent of ASP(+) uptake was inhibited by L-carnitine, an OCTN2-carried zwitterion. The uptake was pH dependent, with approximately 3-fold lower rates at acidic (pH 5.7) than at alkaline (pH 8.2) extracellular pH. Albuterol and formoterol inhibited ASP(+) uptake, suggesting that all these molecules are carried by the same transport mechanism. These findings demonstrate the existence and functional role of a pH-dependent organic cation uptake machinery, namely OCTN1 and OCTN2, in human airway epithelia. We suggest that epithelial OCTN1/2 are involved in the delivery of inhaled cationic bronchodilators to the airway tissue.

Figure 1.

Comparison of organic cation transporter mRNA expression levels in human tissues and cells. Relative mRNA expression levels of the membrane potential sensitive organic cation transporters (OCT1, OCT2, and OCT3) and the pH-dependent organic cation/carnitine transporters (OCTN1 and OCTN2) were measured using quantitative RT-PCR. Tracheas and large bronchi (⩾ third generation) were obtained from human donors without preexisting lung disease (n of donors = 4), and from patients with cystic fibrosis (ΔF508/ΔF508 homozygote and ΔF508/D1152H heterozygote) (n of donors = 2). RNA was extracted from scraped airway epithelium and/or cells cultured and re-differentiated at the ALI. Placenta, kidney, and liver RNA samples were obtained from a single donor subject. Expression data are indicated relative to the sample (calibrator) with the highest expression level of the respective gene: liver for OCT1, kidney for OCT2, OCTN1, and OCTN2, and placenta for OCT3, respectively. Bars indicate the average of triplicate quantitative RT-PCR measurements. Error bars indicate SEM for samples obtained from four different human donors. ND = not detectable level.

Figure 2.

Immunofluorescence analysis reveals polarized expression of the pH-dependent organic cation transporter OCTN1 and OCTN2 in human airways. After autofluorescence reduction and antigen retrieval (see Materials and Methods), human trachea and lung parenchyma sections were incubated with 5 μg/ml affinity-purified goat IgG against human OCTN1 (A–D) and OCTN2 (E–H), or nonimmune goat IgG as a negative control (I–L) followed by AlexaFluor 488–coupled rabbit anti-goat IgG. Sections were mounted in a reagent containing DAPI to stain nuclei (blue). OCTN1 (green) is primarily localized to the apical surface of tracheal epithelia (B), whereas less staining is seen in alveolar epithelia (D). Inflammatory cells in the trachea and the alveoli reveal OCTN1 immunoreactivity. OCTN2 (green) is also localized to the apical surface of airway epithelial cells (F). OCTN2 immunoreactivity is largely associated with the surface of the alveolar epithelia in the lung parenchyma (G). Figure shows bright field (A, C, E, G, I, K) and fluorescence images (B, D, F, H, J, L).

Figure 3.

Immunolocalization of OCTN1 and OCTN2 proteins in human airway epithelial cells grown and re-differentiated in ALI cultures. To identify cellular localization of the pH-dependent organic cation transporters, epithelial cultures were double-labeled with antibodies to OCTN1, OCTN2, or nonimmune IgG (negative control) (green) and acetylated tubulin (red) for analysis with confocal laser scanning microscopy. Samples were mounted in a reagent containing DAPI to stain nuclei (blue). Images are z-axis reconstructions of OCTN1 (A), OCTN2 (B), and acetylated tubulin expression (D–F); overlay z-axis reconstructions (G–I); and overlay confocal images at the level of the cilia (J–L). Images demonstrate OCTN1 and OCTN2 expression in the apical/subapical portion of the cells. Overlay images reveal co-localization of OCTN2 and acetylated tubulin (H and K) indicating OCTN2 protein expression in cilia of airway epithelial cells.

Figure 4.

Uptake measurements with the cationic fluorophore ASP⁺ indicate a carrier-mediated mechanism for cationic drug uptake in human airway epithelial cells. Unexposed primary airway epithelial cells (A) and cells exposed to 10 μM ASP⁺ for 15 min at 37°C (B). Images show ASP⁺ (red) and DAPI nuclear stain fluorescence (blue). (C) Time course of fluorescence (F) after exposing the cells to 10 μM ASP⁺ at 37°C. (D) Effect of temperature on fluorescence after exposing the cells to ASP⁺ for 15 min (E) Temperature-sensitive (saturable) uptake of ASP⁺ was calculated as the difference between the fluorescence after exposure to ASP⁺ at 37°C and 4°C. (F) Eadie-Hofstee plot: the single straight line indicates that a single organic cation uptake system exists in airway epithelial cells. Results represent mean ± SEM (n = 3 or 4 experiments).

Figure 5.

Pharmacologic evidence that the ASP⁺ uptake mechanism carries both organic cations and L-carnitine into human airway epithelial cells. (A) Effect of extracellular Na⁺ on ASP⁺ uptake. For the Na⁺-free experiment, NaCl was replaced by an equimolar concentration N-methyl-D-glucamine. (B–D) Effect of organic cation transporter inhibitors, including tetraethylammonium (TEA⁺), corticosterone, L-carnitine, and ergothioneine on ASP⁺ uptake. Cells were incubated with 10 μM ASP⁺ for 15 min at 37°C in all experiments in the presence or absence of the inhibitors. Control was calculated as the temperature-sensitive (saturable) uptake of ASP⁺. Results represent mean ± SEM (n = 3 or 4 experiments). *P < 0.05 versus control.

Figure 6.

Effects of albuterol, formoterol, and salmeterol on ASP⁺ uptake into human airway epithelial cells. ASP⁺ uptake was measured by exposing the cells to 10 μM ASP⁺ for 15 min in the presence or absence of 0.1 mM and 0.5 mM albuterol (A), formoterol (B), or salmeterol (C) at 37°C. Control was calculated as the temperature-sensitive (saturable) uptake of ASP⁺. Results represent mean ± SEM (n = 3 or 4 experiments). *P < 0.05 versus control.

Figure 7.

Effect of extracellular pH on organic cation uptake by human airway epithelial cells. ASP⁺ uptake was measured by exposing the cells to 10 μM ASP⁺ for 15 min at 37°C in an incubation medium adjusted to different pH levels. Control was calculated as the temperature-sensitive (saturable) uptake of ASP⁺. Results represent mean ± SEM (n = 3 or 4 experiments). *P < 0.05 versus control.