Effects of brefeldin-A upon the β-adrenoceptor-mediated control of apical Na+ conductance (GNa) in rat fetal distal lung epithelial (FDLE) cells

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  • Effects of brefeldin-A upon the β-adrenoceptor-mediated control of apical Na+ conductance (GNa) in rat fetal distal lung epithelial (FDLE) cells

University of Leeds (2002) J Physiol 544P, S112

Communications: Effects of brefeldin-A upon the β-adrenoceptor-mediated control of apical Na+ conductance (GNa) in rat fetal distal lung epithelial (FDLE) cells

S.J. Ramminger, R.E. Olver and S.M. Wilson

Lung Membrane Transport Group, Tayside Institute of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK

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If breathing is to be established at birth then the liquid secreted into the lung lumen during fetal life must first be removed from the potential air spaces. This occurs during the final stages of pregnancy and is driven by the β-adrenoceptor-mediated transport of Na+ out of the alveolar space (Olver et al. 1986). It has been suggested that this stimulation of Na+ transport involves the insertion of additional Na+ channels into the apical plasma membrane by an exocytotic mechanism that is sensitive to brefeldin A (Ito et al. 1997). Recent work from this laboratory has shown that β-adrenoceptor agonists do increase GNa in FDLE cells (Collett et al. 2002) and so we now explore the effects of brefeldin-A upon this response.

These data confirm (Collett et al. 2002) that isoprenaline-evoked increase in ISC (basal, 7.9 ± 0.7 mA cm-2; post-isoprenaline, 11.2 ± 0.9 mA cm-2, P < 0.02) is normally associated with a rise in GNa (Fig. 1). Exposing unstimulated cells to brefeldin-A (1 mM) caused a barely discernible rise in ISC that developed over a period of ~5 min. The current then declined slowly, reaching a value that was 88.7 ± 3.6 % of its initial level (P < 0.05) after 30 min. Application of isoprenaline to brefeldin A-treated cells did not elicit a discernible change in ISC. Data obtained after permeabilisation showed that this drug causes a fall in GNa and blocks the isoprenaline-evoked increase in conductance (Fig. 1). Isoprenaline thus causes a brefeldin-A-sensitive rise in GNa in FDLE cells (Ito et al. 1997; Collett et al. 2002).

The authors thank The Wellcome TRust and Tenovus (Scotland) for their financial support.

All procedures accord with current UK legislation.

Figure 1. Time-mated pregnant rats were anaesthetised on the 20th day of gestation, their fetuses delivered by Caesarean section and immediately decapitated and the donor rats humanely killed without regaining consciousness. FDLE cells were then isolated from the fetal lung tissue as described previously (Collett et al. 2002). This acquisition of fetal lung tissue accords with current UK legislation. The cells were grown to confluence (~48 h) on Snapwell membranes (Costar) which were then mounted in Ussing chambers and ISC measured. The cells were then basolaterally permeabilised (75 mM nystatin) so that GNa could be estimated by measuring the amiloride-sensitive current evoked by imposing an inwardly directed Na+ gradient upon the cells (Collett et al. 2002). Such experiments were undertaken using control and isoprenaline-stimulated cells that had either been maintained under control conditions or exposed to brefeldin-A (1 mM) for 30 min. Data are presented as means ± S.E.M. (n = 6). ***P < 0.001 vs. data derived from the unstimulated cells; †P < 0.05 vs. data derived from unstimulated control cells (Student's paired t test).\"
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Figure 1. Time-mated pregnant rats were anaesthetised on the 20th day of gestation, their fetuses delivered by Caesarean section and immediately decapitated and the donor rats humanely killed without regaining consciousness. FDLE cells were then isolated from the fetal lung tissue as described previously (Collett et al. 2002). This acquisition of fetal lung tissue accords with current UK legislation. The cells were grown to confluence (~48 h) on Snapwell membranes (Costar) which were then mounted in Ussing chambers and ISC measured. The cells were then basolaterally permeabilised (75 mM nystatin) so that GNa could be estimated by measuring the amiloride-sensitive current evoked by imposing an inwardly directed Na+ gradient upon the cells (Collett et al. 2002). Such experiments were undertaken using control and isoprenaline-stimulated cells that had either been maintained under control conditions or exposed to brefeldin-A (1 mM) for 30 min. Data are presented as means &plusmn; S.E.M. (n = 6). ***P &lt; 0.001 vs. data derived from the unstimulated cells; &#8224;P &lt; 0.05 vs. data derived from unstimulated control cells (Student's paired t test).\"

Where applicable, experiments conform with Society ethical requirements.

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