Richard E. Olver

Oxygen‐evoked Na+ transport in rat fetal distal lung epithelial cells

1 Monolayer cultures of rat fetal distal lung epithelial (FDLE) cells generated larger spontaneous short circuit currents (ISC) when maintained (48 h) at neonatal alveolar PO2 (100 mmHg) than at fetal PO2 (23 mmHg). When cells were shifted between these atmospheres in order to impose a rise in PO2 equivalent to that seen at birth, no rise in ISC was seen after 6 h but the response was fully established by 24 h. 2 Studies of basolaterally permeabilised cells revealed a small rise in apical Na+ conductance (GNa) 6 h after PO2 was raised but no further change had occurred by 24 h. A substantial rise was, however, seen after 48 h. 3 Reporter gene assays showed that no activation of the α‐ENaC (epithelial Na+ channel α‐subunit) promoter was discernible 24 h after PO2 was raised but increased transcriptional activity was seen at 48 h. 4 Studies of apically permeabilised cells showed that a small rise in Na+ pump capacity was evident 6 h after PO2 was raised and, in common with the rise in ISC, this effect was fully established by 24 h. The rise in ISC thus develops 6‐24 h after PO2 is raised and is due, primarily, to increased Na+ pump capacity. 5 The increase in GNa thus coincides with activation of the α‐ENaC promoter but these effects occur after the rise in ISC is fully established and so cannot underlie this physiological response. The increased transcription may be an adaptation to increased Na+ transport and not its cause.

The influence of mode of delivery, hormonal status and postnatal O2 environment on epithelial sodium channel (ENaC) expression in perinatal guinea-pig lung.

We have studied factors that potentially modulate the expression of mRNA coding for subunits of the amiloride‐sensitive sodium channel, αENaC and βENaC, in lungs of vaginally and Caesarean (CS)‐delivered late gestation fetal guinea‐pigs. Expression of αENaC and βENaC mRNAs was developmentally regulated in the late gestation fetus, reaching peak levels at term (68 days post conception, PC) and postnatally, respectively. In animals delivered by CS at 65 days PC and term, αENaC mRNA expression was significantly increased by day 1 post partum, reaching levels greater than those normally achieved in vaginally delivered animals at term. In contrast, βENaC mRNA levels remained significantly lower postnatally in animals delivered by CS at 65 days PC compared with those in vaginally and CS‐delivered animals at term. Plasma cortisol and total triiodothyronine (T3) levels increased towards term, were higher 1 day after vaginal delivery but declined towards pre‐term levels by day 3. Cortisol levels also increased rapidly in the CS‐delivered animals, reaching levels similar to those in vaginally delivered animals at day 1. Plasma T3 levels at days 1 and 3 were significantly lower in animals delivered by CS at 65 days PC. The increase in αENaC mRNA paralleled the increase in plasma cortisol after delivery, but not T3, and inhibition of cortisol synthesis with 2‐methyl‐1,2‐di‐3‐pyridyl‐1‐propanone (metyrapone) after CS delivery suppressed the increase in αENaC mRNA expression. Concomitant with the increase in αENaC mRNA expression after CS delivery at 65 days PC was an increase in the amiloride‐blockable component of lung fluid clearance by day 3 postnatally. We conclude that in late gestation guinea‐pigs delivered by CS there is a significant increase in lung αENaC expression postnatally, which is mediated, in part, by the postnatal rise in cortisol at delivery. This in turn leads to an increase in amiloride‐sensitive lung fluid clearance, which is unrelated to labour.

The effects of PO2 upon transepithelial ion transport in fetal rat distal lung epithelial cells

1 Isolated rat fetal distal lung epithelial (FDLE) cells were cultured (for 48 h) at PO2 levels between 23 and 142 mmHg. Higher PO2 levels between 23 and 142 mmHg. Higher PO2 was associated with increased short circuit current (ISC) and increased abundance of the Na+ channel protein α‐ENaC. PO2 had no effect upon ISC remaining after apical application of amiloride (10 μM). 2 Studies of cells maintained (for 48 h) at PO2 levels of 23 mmHg or 100 mmHg, and subsequently nystatin permeabilized (50 μM), showed that high PO2 increased Na+ pump capacity. This response was apparent 24 h after PO2 was raised whilst it took 48 h for the rise in ISC seen in intact cells to become fully established. Both parameters were unaffected by raising PO2 for only 30 min. 3 Basolateral application of isoprenaline (10 μM) did not affect ISC in cells maintained at 23 mmHg but evoked progressively larger responses at higher PO2. The response seen at 142 mmHg was larger than at 100 mmHg, the normal physiological alveolar PO2. 4 Isoprenaline had no effect on Na+ pump capacity at PO2 levels of 23 mmHg or 100 mmHg, but stimulated Na+ extrusion at 142 mmHg. Increasing PO2 above normal physiological levels thus allows the Na+ pump to be controlled by isoprenaline. This may explain the enhanced sensitivity to isoprenaline seen under these slightly hyperoxic conditions. 5 Changes in PO2 mimicking those occurring at birth thus exert profound influence over Na+ transport in FDLE cells and the Na+ pump could be an important locus at which this control is exercised.

Systematic review of antistaphylococcal antibiotic therapy in cystic fibrosis

Specific interventions included in the review The following antibiotics were used either alone or in combination: trimethoprim; sulphamethoxazole; cefadroxil; dicloxacillin; cephalexin; flucloxacillin; cloxacillin; ampicillin; amoxycillin (with and without clavulanic acid); penicillin V; erythromycin; cotrimoxazole; cephalosporin; ciprofloxacin; fusidic acid; lincomycin; chloramphenicol; novobiocin; carbenicillin; gentamycin; clindamycin; oxacillin; rifampicin; methicillin; cephuroxime; and macrolide. Drugs were given either continuously or intermittently using oral, intravenous and inhalation administration methods.

P2Y2 receptor-mediated inhibition of ion transport in distal lung epithelial cells

Rat foetal distal lung epithelial cells were plated onto permeable supports where they became integrated into epithelial sheets that spontaneously generated short circuit current (ISC). Apical ATP (100 μM) evoked a transient fall in ISC that was followed by a rise to a clear peak which, in turn, was succeeded by a slowly developing decline to a value below control. Apical UTP evoked an essentially identical response. UDP and ADP were ineffective whilst ATP had no effect when added to the basolateral solution. These effects thus appear to be mediated by apical P2Y2 receptors. The rising phase of the responses to ATP/UTP was selectively inhibited by anion transport inhibitors but persisted in the presence of amiloride, which abolished the inhibitory effects of both nucleotides. Thus, apical nucleotides appear to evoke a transient stimulation of anion secretion and sustained inhibition of Na+ absorption. Basolateral isoprenaline (10 μM) elicited a rise in ISC but subsequent addition of apical ATP reversed this effect. Conversely, isoprenaline restored ISC to its basal level following stimulation with ATP. Apical P2Y2 receptors and basolateral β‐adrenoceptors thus allow their respective agonists to exert mutually opposing effects on ISC.

Regulation of intracellular pH in Calu-3 human airway cells

The Calu‐3 human cell line exhibits features of submucosal gland serous cells and secretes HCO3−. The aim of this study was to identify the HCO3− transporters present in these cells by studying their role in the regulation of intracellular pH (pHi). Calu‐3 cells were grown on coverslips, loaded with the pH‐sensitive fluorescent dye BCECF, and their fluorescence intensity monitored as an indication of pHi. Cells were acidified with NH4Cl (25 mm, 1 min) and pHi recovery recorded. In the absence of HCO3−, initial recovery was 0.208 ± 0.016 pH units min−1(n = 37). This was almost abolished by removal of extracellular Na+ and by amiloride (1 mm), consistent with the activity of a Na+‐H+ exchanger (NHE). In the presence of HCO3− and CO2, recovery (0.156 ± 0.018 pH units min−1) was abolished (reduced by 91.8 ± 6.7 %, n = 7) by removal of Na+ but only attenuated (by 63.3 ± 5.8 %, n = 9) by amiloride. 4,4‐Dinitrostilbene‐2,2‐disulfonic acid (DNDS) inhibited recovery by 45.8 ± 5.0 % (n = 7). The amiloride‐insensitive recovery was insensitive to changes in membrane potential, as confirmed by direct microelectrode measurements, brought about by changing extracellular [K+] in the presence of either valinomycin or the K+ channel opener 1‐EBIO. In addition, forskolin (10 μm), which activates the cystic fibrosis transmembrane conductance regulator Cl− conductance in these cells and depolarises the cell membrane, had no effect on recovery. Removal of extracellular Cl− trebled pHi recovery rates, suggesting that an electroneutral, DNDS‐sensitive, Cl−‐HCO3− exchanger together with a NHE may be involved in pHi regulation and HCO3− secretion in these cells. RT‐PCR detected the expression of the electrogenic Na+‐HCO3− cotransporter NBC1 and the Cl−‐HCO3− exchanger (AE2) but not the electroneutral Na+‐HCO3− cotransporter NBCn1.

Developmental regulation of lumenal lung fluid and electrolyte transport

In the fetus, there is a net secretion of liquid (LL) by the lung as a result of active transport of chloride ions. The rate of secretion and the resulting volume of LL are vital for normal lung growth but how volume is sensed and how secretion may be regulated are still unknown. Towards term under the influence of thyroid and adrenocorticoid hormones, the epithelial sodium channel (ENaC) is increasingly expressed in the pulmonary epithelium. Adrenaline released by the fetus during labour activates ENaC and produces rapid absorption of liquid in preparation for air breathing; absence of ENaC is incompatible with survival. There may be other mechanisms involved in aiding liquid clearance including changes in epithelial permeability, an effect of oxygen on both ENaC and Na/K ATPase and perhaps the influence of additional hormones on ENaC activity. Some time after birth there are further developmental changes with the appearance of other cation channels (CNG1 and perhaps NSCC) which contribute to the liquid absorptive side of the balance existing across the epithelium between secretion and absorption to produce essentially almost no net liquid movement in the postnatal lung. The evidence for these processes is discussed and areas of uncertainty indicated.

Sodium-proton exchange across the apical membrane of the alveolar type II cell of the fetal sheep.

In order to detect and characterise Na(+)-H+ countertransport in the fetal lung epithelium we have studied under a variety of conditions the effect of an outward facing H+ gradient on Na+ uptake into purified apical membrane vesicles prepared from alveolar type II cells. Kinetic analysis of the data reveals both a diffusional and a saturable component of total Na+ uptake. Evidence for the presence of a Na(+)-H+ exchanger is demonstrated by (1) stimulation of Na+ uptake by proton loading of vesicles both in the presence and absence of chemical voltage clamping; (2) saturation kinetics with respect to external Na+ with a Km of 16 mM and a Vmax of 2.1 nmol/mg protein per min; (3) amiloride inhibition of Na+ uptake driven by pH gradient. We conclude that although diffusion may be the major component of total Na+ uptake at physiological external Na+ concentration, Na(+)-H+ countertransport provides a possible mechanism for the acidification of fetal lung liquid in-vivo in addition to its established role in intracellular pH and volume regulation.

Differential effects of UTP and ATP on ion transport in porcine tracheal epithelium

Isolated segments of porcine tracheal epithelium were mounted in Ussing chambers, current required to maintain transepithelial potential difference at 0 mV (short circuit current, ISC) was monitored and effects of nucleotides upon ISC were studied. Mucosal UTP (100 μM) evoked a transient rise in ISC that was followed by a sustained fall below basal ISC maintained for 30 min. Mucosal ATP (100 μM) also stimulated a transient rise in ISC but in contrast to UTP did not inhibit basal ISC. Submucosal UTP and ATP both transiently increased ISC. UTP‐prestimulated epithelia were refractory to ATP but prestimulation with ATP did not abolish the response to UTP. The epithelia thus appear to express two populations of apical receptors allowing nucleotides to modulate ISC. The UTP‐induced rise was reduced by pretreatment with either bumetanide (100 μM), diphenylamin‐2‐carboxylic acid (DPC, 1 mM), or Cl− and HCO3−‐free solution whilst the fall was abolished by amiloride pretreatment. Thapsigargin (0.3 μM) abolished the UTP‐induced increase in ISC but not the subsequent decrease. Staurosporine (0.1 μM) inhibited basal ISC and blocked UTP‐induced inhibition of ISC. Inhibitors of either protein kinase C (PKC) (D‐erythro sphingosine) or PKA (H89) had no effect. This study suggests that UTP stimulates Cl− secretion and inhibits basal Na+ absorption. ATP has a similar stimulatory effect, which may be mediated by activation of P2Y2 receptors and an increase in [Ca2+]in, but no inhibitory effect, which is likely mediated by activation of a pyrimidine receptor and possible inhibition of a protein kinase other than PKC or PKA.

The regulation of selective and nonselective Na+ conductances in H441 human airway epithelial cells.

Analysis of membrane currents recorded from hormone-deprived H441 cells showed that the membrane potential (V(m)) in single cells (approximately -80 mV) was unaffected by lowering [Na+]o or [Cl(-)]o, indicating that cellular Na+ and Cl(-) conductances (GNa and GCl, respectively) are negligible. Although insulin (20 nM, approximately 24 h) and dexamethasone (0.2 microM, approximately 24 h) both depolarized Vm by approximately 20 mV, the response to insulin reflected a rise in GCl mediated via phosphatidylinositol 3-kinase (PI3K) whereas dexamethasone acted by inducing a serum- and glucocorticoid-regulated kinase 1 (SGK1)-dependent rise in GNa. Although insulin stimulation/PI3K-P110 alpha expression did not directly increase GNa, these maneuvers augmented the dexamethasone-induced conductance. The glucocorticoid/SGK1-induced GNa in single cells discriminated poorly between Na+ and K+ (PNa/PK approximately 0.6), was insensitive to amiloride (1 mM), but was partially blocked by LaCl3 (La3+; 1 mM, approximately 80%), pimozide (0.1 mM, approximately 40%), and dichlorobenzamil (15 microM, approximately 15%). Cells growing as small groups, on the other hand, expressed an amiloride-sensitive (10 microM), selective GNa that displayed the same pattern of hormonal regulation as the nonselective conductance in single cells. These data therefore 1) confirm that H441 cells can express selective or nonselective GNa (14, 48), 2) show that these conductances are both induced by glucocorticoids/SGK1 and subject to PI3K-dependent regulation, and 3) establish that cell-cell contact is vitally important to the development of Na+ selectivity and amiloride sensitivity.