Airway epithelial cells respond to changes in the osmolarity of airway surface liquid. In 16HBE14o- cells, a human bronchial epithelial (HBE) cell line, regulatory volume decrease (RVD) requires the activation of a Ca²⁺-dependent potassium channel, triggered by the influx of Ca²⁺ via a gadolinium (Gd³⁺)-sensitive, swelling-activated cation channel (Fernández-Fernández et al. 2002) of unknown molecular identity. TRPV4, a member of the transient receptor potential family of ion channels, which is osmosensitive and inhibitable by Gd³⁺, has been recently described (Strotman et al. 2000). The aim of the present work was to characterize the functional expression of TRPV4 channels in HBE cells.

Expression of TRPV4 in HBE cells was assessed using RT-PCR and Western-blot techniques. Full-length cDNA amplification indicated two isoforms of which one lacked exon 6, encoding part of the third ankyrin repeat and one protein kinase C phosphorylation site. Western-blot analysis using a polyclonal antibody against the C terminus of TRPV revealed a band of ~120 kDa.

The activity of TRPV4 channels in HBE cells was examined measuring intracellular Ca²⁺ with Fura-2 fluorescent dye. Changes in Ca²⁺ were measured as the ratio of emission fluorescence intensities obtained following excitation with 340 nm and 380 nm wavelengths in cells loaded with 2 µM Fura-2. Cells challenged with a 28 % hypotonic solution responded with a transient increase in fluorescence ratio from 0.35 ± 0.01 to 0.5 ± 0.03 (n = 3, means ± S.E.M.). The phorbol ester analogue, 4α-phorbol 12,13-didecanoate (4α-PDD, 1 µM), known to activate TRPV4 (Watanabe et al. 2002) induced a larger increase in the fluorescence ratio from 0.35 ± 0.01 to 1.18 ± 0.1 (n = 3). In the presence of 100 µM Gd³⁺, the fluorescence ratio in response to hypotonic shock and 4α-PDD changed from 0.34 ± 0.02 to 0.37 ± 0.02 and from 0.4 ± 0.006 to 0.54 ± 0.02, respectively (n = 4). The electrophysiological activity of TRPV4 channels was also recorded in HBE cells using a NaCl-rich bathing solution containing 2 mM CaCl₂ and a Ca²⁺-free intracellular N-methyl-D-glucamine chloride solution. A reversible increase in cationic current density from -3.21 ± 2.4 pA pF^-1 (control) to -27.3 ± 10.6 pA pF^-1 (n = 4) was recorded (at -120 mV) upon addition of 1 µM 4α-PDD.

Our data provide the first functional and molecular identification of endogenously expressed TRPV4 channels in human airway epithelia.

This work was supported by Ministerio de Ciencia y Tecnolog’a (España) and Distinció de la Generalitat de Catalunya.