Raising the P_O2 to which alveolar epithelial cells are exposed increases transepithelial amiloride-sensitive sodium transport. These changes are associated with an increase in apical conductance, sodium channel α-subunit (αENaC) mRNA levels and transcriptional activity of the αENaC gene (Baines et al. 2001). By making deletion constructs of the αENaC gene I have explored further the regulation of transcriptional activity of this gene by changes in P_O2. 5Ì Promotor regions of the αENaC gene (containing a glucocorticoid response element (GRE) and several transcription factor binding sites such as NF-κB, AP2 and SP1, were cloned into pGL3 luciferase reporter vector and transiently transfected into A549 lung epithelial cells maintained in D-MEM supplemented with 10 % charcoal-stripped serum. Following an overnight incubation (18 h) at a P_O2 of either 23 or 142 mmHg, cells were shifted either to a P_O2 of 142 or 23 mmHg for 6 or 24 h. Luciferase activity of the cell lysates was measured by luminometry and is directly related to the promoter activity of the αENaC construct. All activities were compared with activity of a promoterless construct (to give measure of minimum activity – pGL3.basic) and a constitutively active promoter construct (to give measure of maximal activity – pGL3.control). Transfection efficiency was normalised by co-transfection with pSV-β galactosidase expression vector.

Transcriptional activity of constructs containing either the 5Ì (E2.2, E1.9, E1.4) and/or the 3Ì transcriptional start site (E2.2, E0.8) was significantly higher than pGL3.basic (luciferase activity construct/pGL3 basic, E2.2, 5.9 ± 1.4; E1.9, 49 ± 16; E1.4, 37 ± 18; E0.8, 13 ± 6, P < 0.05, n = 4) showing that all the promoter regions studied could actively drive transcription from either start site. In stripped serum, changing P_O2 evoked no significant changes in promoter activity of any of the constructs. Dexamethasone (Dex, 100 µM) evoked a robust increase (33.6 ± 8-fold at 24 h) in activity of the GRE-containing construct. Shifting P_O2 from either 23 to 142 mmHg or from 142 to 23 mmHg for 6 h significantly inhibited Dex-induced activation compared with cells maintained at 23 mmHg (13 ± 1.3 to 7.2 ± 1.1 and 8.5 ± 1.4 to 6.1 ± 1.1, respectively, P < 0.05, n = 4); this inhibitory effect of P_O2 was transient and no longer significant at 24 h. These data show that moving from hypoxia to normoxia, or vice versa, can transiently suppress the glucocorticoid-driven transcription of αENaC and suggests that environmental P_O2 may have its most important regulatory role in situations where GRE is maximally driving transcription (e.g. around the time of normal term delivery).

Baines, D.L., Ramminger, S.J., Collett, A., Haddad, J.J., Best, O.G., Land, S.C., Olver, R.E. & Wilson S.M. (2001). J. Physiol. 532, 105-113. abstract