Nerve growth factor reduces amiloride-sensitive Na+ transport in human airway epithelial cells

Abstract

Nerve growth factor (NGF) is overexpressed in patients with inflammatory lung diseases, including virus infections. Airway surface liquid (ASL), which is regulated by epithelial cell ion transport, is essential for normal lung function. No information is available regarding the effect of NGF on ion transport of airway epithelium. To investigate whether NGF can affect ion transport, human primary air-interface cultured epithelial cells were placed in Ussing chambers to obtain transepithelial voltage (-7.1 ± 3.4 mV), short-circuit current (Isc, 5.9 ± 1.0 μA), and transepithelial resistance (750 Ω·cm(2)), and to measure responses to ion transport inhibitors. Amiloride (apical, 3.5 × 10(-5) mol/L) decreased Isc by 55.3%. Apically applied NGF (1 ng/mL) reduced Isc by 5.3% in 5 min; basolaterally applied NGF had no effect. The response to amiloride was reduced (41.6%) in the presence of NGF. K-252a (10 nmol/L, apical) did not itself affect Na(+) transport, but it attenuated the NGF-induced reduction in Na(+) transport, indicating the participation of the trkA receptor in the NGF-induced reduction in Na(+) transport. PD-98059 (30 μmol/L, apical and basolateral) did not itself affect Na(+) transport, but attenuated the NGF-induced reduction in Na(+) transport, indicating that trkA activated the Erk 1/2 signaling cascade. NGF stimulated phosphorylation of Erk 1/2 and the β-subunit of ENaC. K-252a and PD-98059 inhibited these responses. NGF had no effect on Isc in the presence of apical nystatin (50 μmol/L). These results indicate that NGF inhibits Na(+) transport through a trkA-Erk 1/2-activated signaling pathway linked to ENaC phosphorylation.

Keywords: Airway epithelium; electrophysiology; ion transport; lung; nerve growth factor.

Figure 1.

Time course of the development of R_t in air–liquid interface (ALI)‐cultured normal human bronchial epithelial cells (NHBE). R_t was monitored over 30 days on ALI. NHBE cells generated and maintained high resistance after 10 days on ALI culture (n = 1 donor, 12 replicates).

Figure 2.

Confirmation of normal human bronchial epithelial cell (NHBE) differentiation. The differentiation of NHBE cells was confirmed through several imaging techniques. (A) Hematoxylin and eosin staining revealed a pseudo‐stratified epithelium with the presence of cilia, (B) alcian blue staining confirmed mucus production, and (C) scanning electron microscopy revealed the presence of cilia on the apical surface. These structures were confirmed to be cilia through the use of (D) transmission electron microscopy, which revealed the presence of a 9 + 2 doublet of microtubules, and (E) immunofluorescence for β‐tubulin.

Figure 3.

Representative I_sc tracings of responses to known ion transport inhibitors. Once I_sc stabilized, responses to amiloride (3.5 × 10⁻⁵ mol/L), NPPB (10⁻⁴ mol/L), and ouabain (10⁻⁴ mol/L) were generated in the (A) absence or (B) presence of 1 ng/mL nerve growth factor (NGF).

Figure 4.

Effect of nerve growth factor (NGF) on ion transport. Responses to (A) amiloride, (B) NPPB, and (C) ouabain were calculated as a percent change from baseline (4 donors). NGF significantly reduced the amiloride response (P = 0.0127).

Figure 5.

Effect of nerve growth factor (NGF) on amiloride‐sensitive Na⁺ transport. Normal human bronchial epithelial cells cultured from four donors demonstrated consistent reductions in Na⁺ transport in the presence of 1 ng/mL NGF.

Figure 6.

Effect of nerve growth factor (NGF) on responses to apically applied amiloride after 30 min. Although the response to amiloride was reduced in the presence of NGF, this reduction was not significant. n = 4.

Figure 7.

Effect of nerve growth factor (NGF)‐induced trkA activation on amiloride‐sensitive Na⁺ transport. Cells were either incubated apically with the nonspecific tyrosine kinase inhibitor, K‐252a, or DMSO for 30 min prior to generating responses to (A) NGF, (B) amiloride, (C) NPPB, and (D) ouabain in the absence or presence of NGF. Incubation with K‐252a significantly attenuated both the (A) NGF response (P = 0.04) and (B) the NGF induced reduction in amiloride‐sensitive Na⁺ (*P = 0.002; ^#P = 0.041). DMSO n = 4; all other groups n = 6.

Figure 8.

The involvement of the trkA downstream signaling pathway, Erk 1/2, in the nerve growth factor (NGF)‐induced reduction in amiloride‐sensitive Na⁺ transport. Cells were either incubated apically and basolaterally with the specific Erk 1/2 inhibitor, PD‐98059, or DMSO for 30 min prior to generating responses to (A) NGF, (B) amiloride, (C) NPPB, and (D) ouabain in the absence or presence of NGF. Incubation with PD‐98059 significantly attenuated both the (A) NGF response (P = 0.001) and (B) the NGF induced reduction in amiloride‐sensitive Na⁺ transport (*P = 0.002; ^#P = 0.012). DMSO n = 4; all other groups n = 6.

Figure 9.

The effect of nerve growth factor (NGF) in epithelial cells permeabilized apically with nystatin. Cells were placed into Ussing chambers and allowed to equilibrate prior to adding nystatin (50 μmol/L) to the apical chamber. Nystatin caused a large increase in I_sc. Responses to ouabain were generated in the (A) absence (vehicle control – modified Krebs‐Henseleit solution [MKHS]) or (B) presence of 1 ng/mL NGF. NGF did not elicit bioelectric responses when applied apically to the permeabilized cells, and did not alter Na⁺/K⁺‐ATPase activity as there were no differences in the response to ouabain. Control n = 6, NGF n = 4. Scale bar = 10 min.

Figure 10.

Effect of prolonged incubation with nerve growth factor (NGF) on ion transport. Responses to known ion transport inhibitors were generated after incubating cells 24 h (A–C) and 48 h (D–F) with 1 ng/mL NGF. There were no difference in response to amiloride (A and D), NPPB (B and E), and ouabain (C and F) between control or NGF treated cells following either a 24‐ or 48‐h incubation (2 donors).

Figure 11.

Western blots showing the effects of nerve growth factor (NGF) on Erk 1/2 activation and ENaC phosphorylation. Cells were incubated apically with modified Krebs‐Henseleit solution (MKHS; control) or 1 ng/ml NGF in MKHS for 5 min. (A) NGF activated the Erk 1/2 signaling pathway. This activation was inhibited by K‐252a and PD‐98059. (B) NGF‐mediated activation of Erk 1/2 resulted in ENaC phosphorylation, and was inhibited with K‐252a and PD‐98059. (C) Representative blots for Erk 1/2 (42 and 44 kDa), phosphorylated Erk 1/2 (P‐Erk; 44 and 45 kDa), phosphorylated ENaC (76 kDa), and the loading control, β‐actin (47 kDa). n = 4. *P < 0.05.

Figure 12.

Western blots showing the effect of nerve growth factor (NGF) on β‐ENaC. (A and C) Cells incubated with NGF for 5 min demonstrated a threefold increase in phosphorylated‐β‐ENaC (76 kDa). (B) Blots were stripped and probed for β‐ENaC (75 kDa) and β‐Actin (42 kDa). NGF did not affect β‐ENaC levels. n = 6, *P < 0.05.