Hypobaric hypoxia-related impairment of pulmonary surfactant proteins A and D did not favour Pneumocystis carinii Frenkel 1999 growth in non-immunocompromised rats.

It has been suggested that patients with pulmonary surfactant impairment are more susceptible to Pneumocystis infection than healthy controls. Owing the fact that most patients with pulmonary surfactant impairment also suffer from hypoxia, we explored the effect of intermittent hypobaric hypoxia conditions on the ability of non-immunocompromised rats infected by endotracheal route with P. carinii to clear the infection from their lungs. Control rats, inoculated or not with P. carinii, were maintained in normobaric normoxic conditions, and were submitted or not to dexamethasone administration. It was found that even if hypobaric hypoxia weakened host immune mechanisms and impaired significantly the surfactant composition, mainly of surfactant proteins A and D, these changes were not enough to favour the Pneumocystis growth or to inhibit the clearing of Pneumocystis organisms from the lungs of non-immunocompromised rats. The potential influence of surfactant protein changes on Pneumocystis infection is discussed.

Modulation of alpha-ENaC and alpha1-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells.

cAMP and dexamethasone are known to modulate Na+ transport in epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and dexamethasone modulate the mRNA expression of two key elements of the Na+ transport system in isolated rat alveolar epithelial cells: alpha-, beta-, and gamma-subunits of the epithelial Na+ channel (ENaC) and the alpha1- and beta1-subunits of Na+-K+-ATPase. The cells were treated for up to 48 h with DBcAMP or dexamethasone to assess their long-term impact on the steady-state level of ENaC and Na+-K+-ATPase mRNA. DBcAMP induced a twofold transient increase of alpha-ENaC and alpha1-Na+-K+-ATPase mRNA that peaked after 8 h of treatment. It also upregulated beta- and gamma-ENaC mRNA but not beta1-Na+-K+-ATPase mRNA. Dexamethasone augmented alpha-ENaC mRNA expression 4.4-fold in cells treated for 24 h and also upregulated beta- and gamma-ENaC mRNA. There was a 1.6-fold increase at 8 h of beta1-Na+-K+-ATPase mRNA but no significant modulation of alpha1-Na+-K+-ATPase mRNA expression. Because DBcAMP and dexamethasone did not increase the stability of alpha-ENaC mRNA, we cloned 3.2 kb of the 5' sequences flanking the mouse alpha-ENaC gene to study the impact of DBcAMP and dexamethasone on alpha-ENaC promoter activity. The promoter was able to drive basal expression of the chloramphenicol acetyltransferase (CAT) reporter gene in A549 cells. Dexamethasone increased the activity of the promoter by a factor of 5.9. To complete the study, the physiological effects of DBcAMP and dexamethasone were investigated by measuring transepithelial current in treated and control cells. DBcAMP and dexamethasone modulated transepithelial current with a time course reminiscent of the profile observed for alpha-ENaC mRNA expression. DBcAMP had a greater impact on transepithelial current (2.5-fold increase at 8 h) than dexamethasone (1.8-fold increase at 24 h). These results suggest that modulation of alpha-ENaC and Na+-K+-ATPase gene expression is one of the mechanisms that regulates Na+ transport in alveolar epithelial cells.

Downregulation of epithelial sodium channel (ENaC) by CFTR co-expressed in Xenopus oocytes is independent of Cl- conductance.

Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na+ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl- into the cell and is negligible when Cl- flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na+ current (Iamil) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to -60 mV, stimulating CFTR with 1 mm IBMX reduced Iamil by up to 80%, demonstrating that ENaC is inhibited when Cl- is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of Iamil downregulation and the CFTR-mediated plasma membrane Cl- conductance, suggesting a direct correlation. However, Iamil downregulation was not affected when Cl- flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl- reversal potential (67% +/- 10% inhibition at -20 mV compared to 79% +/- 4% at -60 mV) demonstrating that Iamil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca2+-sensitive photoprotein aequorin showed that Ca2+ is not involved in Iamil downregulation by CFTR, although Ca2+ injection into the cytoplasm did inhibit Iamil. These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca2+ and is independent of the direction and magnitude of Cl- transport across the plasma membrane.

Transcriptional and post-transcriptional regulation of kappaB-controlled genes by pp60v-src.

The CEF-4/9E3 gene is expressed aberrantly in chicken embryo fibroblasts transformed by the Rous sarcoma virus. This aberrant expression is dependent on transcriptional activation and on the stabilization of the CEF-4 mRNA. The characterization of the CEF-4 promoter indicated that three distinct regulatory elements corresponding to an AP-1 binding site, a PRDII/ kappaB domain and a CAAT box are involved in the activation by pp60v-src. Several v-src responsive genes are controlled by AP-1 and members of the Ets family but few appear to be dependent on NF-kappaB. In this study we characterize the expression of genes regulated by NF-kappaB in normal and RSV-transformed CEF. Run-on transcription analysis indicated that pp60v-src induces the transcription of several genes controlled by NF-kappaB but at different levels. While the transcription of CEF-4 was strongly stimulated, that of NF-kappaB1, c-rel, p53 or IkappaB-alpha was activated more modestly by pp60v-src. In addition the CEF-4 mRNA was the only mRNA species to accumulate significantly in transformed CEF. The ectopic expression of RelA or Rel resulted in the stimulation of the transcription of several known targets of NF-kappaB. However, the mRNA for IkappaB-alpha was the only mRNA species to accumulate considerably in the RelA- or Rel-expressing cells. Hence for most kappaB-controlled genes, transcriptional activation was not sufficient to obtain a significant increase in mRNA expression. Likewise, RelA or Rel enhanced the transcription of the CEF-4 gene without a significant accumulation of the CEF-4 mRNA. However, transformation by v-src caused a massive accumulation of the CEF-4 mRNA but not of other mRNA species in the RelA- and Rel-expressing cells. Transient expression assays, run-on transcription and Northern blotting analyses indicated that the effect of pp60v-src on CEF-4 expression was mediated predominantly at the post-transcriptional level in these cells. Therefore transcriptional and post-transcriptional mechanisms determine the restricted pattern of activation of kappaB-controlled genes in RSV-transformed CEF.