Signalling via phosphoinositide-3-kinase (PI3K) / serum - glucocorticoid-inducible kinase 1 (SGK1) is important to the hormonal control of epithelial Na⁺ absorption, and recent work from this laboratory (Watt et al. Brit. J. Pharmacol, in press: 2012) has used a combination of protein biochemistry (Western analysis using phospho-specific antibodies) and electrophysiology (whole cell recording in the perforated patch recording configuration) to explore the role of this signalling pathway in H441 airway epithelial cells. However, the extent to which such established cell lines reproduce the physiology of the intact airway is always open to question, and the aim of the present study was therefore to establish the extent to which these methods can be applied to human airway epithelial cells isolated (with ethical permission) by brushing the nasal turbinate, and maintained in primary culture.

Analysis of extracted protein revealed phosphorylation of protein kinase B at Thr³⁰⁸, a site that is phosphorylation in a PI3K-dependent manner activity, and also revealed phosphorylation of an endogenous SGK1 substate (NDRG1-Thr^356/356/366). It is therefore clear that the PI3K - SGK1 signalling pathway is active under these conditions, and subsequent, electrophysiological studies characterised the membrane currents (I_m) evoked (n = 7) by ramp changes (-112 mV to 86 mV in 1 s) in holding potential. Analysis of currents recorded from single cells exposed to quasi-physiological ionic gradients revealed an I_m - V_Hold relationship that was essentially linear at physiologically relevant potentials but displayed slight outward rectification at depolarized potentials. Regression analysis showed that the resting membrane potential (V_m) was -10.3 ± 2.0 mV. Amiloride (10 mM, 20 - 30 s), a substance known to block ENaC, had no effect upon these currents and thus caused no change in V_m (-10.1 ± 1.6 mV). Lowering [Na⁺]_o to 10 mM (iso-osmotically substituting NMDG⁺) on the other hand, hyperpolarized V_m to -40.6 ± 3.1 mV (P < 0.001, Students t test) and, at least under the present conditions, these cells thus express a Na⁺ conductance (G_Na) that is insensitive to amiloride. To further characterise G_Na, the current that persisted at low Na⁺ was subtracted from the corresponding records of control current in order to isolate the Na⁺-dependent component of I_m (I_Na). Regression analysis (i.e. DI_Na / DV_Hold) of data recorded at negative values of V_Hold showed that the magnitude of G_Na was 13.5 ± 3.9 nS pF^-1, a value which amounted to 53.8 ± 3.4% of the total membrane conductance.