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Lung development requires coordinated signaling between airway and vascular growth, but the link between these processes remains unclear. Mammalian target of rapamycin complex-1 (mTORC1) can amplify hypoxia-inducible factor-1α (HIF-1α) vasculogenic activity through an NH(2)-terminal mTOR binding (TOS) motif. We hypothesized that this mechanism coordinates vasculogenesis with the fibroblast growth factor (FGF)-10/FGF-receptor2b/Spry2 regulator of airway branching. First, we tested if the HIF-1α TOS motif participated in epithelial-mesenchymal vascular signaling. mTORC1 activation by insulin significantly amplified HIF-1α activity at fetal Po(2) (23 mmHg) in human bronchial epithelium (16HBE14o-) and induced vascular traits (Flk1, sprouting) in cocultured human embryonic lung mesenchyme (HEL-12469). This enhanced activation of HIF-1α by mTORC1 was abolished on expression of a HIF-1α (F99A) TOS-mutant and also suppressed vascular differentiation of HEL-12469 cocultures. Next, we determined if vasculogenesis in fetal lung involved regulation of mTORC1 by the FGF-10/FGFR2b/Spry2 pathway. Fetal airway epithelium displayed distinct mTORC1 activity in situ, and its hyperactivation by TSC1(-/-) knockout induced widespread VEGF expression and disaggregation of Tie2-positive vascular bundles. FGF-10-coated beads grafted into fetal lung explants from Tie2-LacZ transgenic mice induced localized vascular differentiation in the peripheral mesenchyme. In rat fetal distal lung epithelial (FDLE) cells cultured at fetal Po(2), FGF-10 induced mTORC1 and amplified HIF-1α activity and VEGF secretion without induction of ERK1/2. This was accompanied by the formation of a complex between Spry2, the cCBL ubiquitin ligase, and the mTOR repressor, TSC2, which abolished GTPase activity directed against Rheb, the G protein inducer of mTORC1. Thus, mTORC1 links HIF-1α-driven vasculogenesis with the FGF-10/FGFR2b/Spry2 airway branching periodicity regulator.

Treating H441 cells with dexamethasone raised the abundance of mRNA encoding the epithelial Na(+) channel alpha- and beta-subunits and increased transepithelial ion transport (measured as short-circuit current, I(sc)) from <4 microA.cm(-2) to 10-20 microA.cm(-2). This dexamethasone-stimulated ion transport was blocked by amiloride analogs with a rank order of potency of benzamil >or= amiloride > EIPA and can thus be attributed to active Na(+) absorption. Studies of apically permeabilized cells showed that this increased transport activity did not reflect a rise in Na(+) pump capacity, whereas studies of basolateral permeabilized cells demonstrated that dexamethasone increased apical Na(+) conductance (G(Na)) from a negligible value to 100-200 microS.cm(-2). Experiments that explored the ionic selectivity of this dexamethasone-induced conductance showed that it was equally permeable to Na(+) and Li(+) and that the permeability to these cations was approximately fourfold greater than to K(+). There was also a small permeability to N-methyl-d-glucammonium, a nominally impermeant cation. Forskolin, an agent that increases cellular cAMP content, caused an approximately 60% increase in I(sc), and measurements made after these cells had been basolaterally permeabilized demonstrated that this response was associated with a rise in G(Na). This cAMP-dependent control over G(Na) was disrupted by brefeldin A, an inhibitor of vesicular trafficking. Dexamethasone thus stimulates Na(+) transport in H441 cells by evoking expression of an amiloride-sensitive apical conductance that displays moderate ionic selectivity and is subject to acute control via a cAMP-dependent pathway.

Redox and ROS regulation of MAPK-mediated TNF-alpha biosynthesis is not well characterized. It was hypothesized that the involvement of the MAPK pathway in regulating LPS-mediated TNF-alpha secretion is redox-dependent, NF-kappaB-sensitive and attenuated by N-acetyl-L-cysteine (NAC) and other antioxidants. In alveolar epithelial cells, LPS induced a time- and dose-dependent phosphorylation of MAPK(p38). This was associated with the activation of MAPK-activated protein kinase, which phosphorylated the small heat-shock protein, Hsp27. MAPK(p38) inhibition (SB-203580) abrogated LPS-induced TNF-alpha production. MAPK(ERK) blockade (PD-98059) attenuated TNF-alpha secretion, an effect synergistically amplified in the presence of SB-203580. Regulation of NF-kappaB by selective inhibitors revealed that this pathway is partially involved in regulating LPS-mediated TNF-alpha secretion. Whereas the proteasome inhibitor, MG-132, had no effect on LPS-mediated TNF-alpha production, CAPE, sulfasalazine and SN-50, a cell-permeant NF-kappaB inhibitor, attenuated but did not abrogate TNF-alpha biosynthesis. LPS up-regulated ROS, an effect abrogated by 4'-hydroxy-3'-methoxy-acetophenone and NAC, which reduced TNF-alpha secretion, induced the accumulation of GSH, reduced the concentration of GSSG, and blockaded the phosphorylation/activation of MAPK(p38) pathway. ROS induced MAPK(p38) phosphorylation and selective antioxidants, including the permeant GSH precursor, gamma-GCE, reduced ROS-dependent MAPK(p38) phosphorylation. These results indicate that the MAPK pathway and MAPK-mediated regulation of TNF-alpha production is redox-dependent, GSH-mediated and requires, at least in part, a NF-kappaB/ROS-sensitive mechanism.

A non-hypoxic, reactive oxygen species (ROS)-sensitive pathway mediating tumor necrosis factor-alpha (TNF-alpha)-dependent regulation of hypoxia-inducible factor-1alpha (HIF-alpha) was investigated in vitro. TNF-alpha mediated the translocation of HIF-1alpha, associated with up-regulating its activity under normoxia. Analysis of the mode of action of TNF-alpha revealed the accumulation of hydrogen peroxide (H2O2), superoxide anion (O(2-.)) and hydroxyl radical (.OH). Antioxidants purported as prototypical scavengers of H2O2 and .OH, attenuated TNF-alpha-induced HIF-1alpha activation, and blockading NADPH-oxidase by scavenging O(2-.) reduced the activity of HIF-1alpha. Inhibition of the mitochondrion complex I abrogated TNF-alpha-dependent activation of HIF-1alpha. Interrupting the respiratory chain reversed the excitatory effect of TNF-alpha on HIF-1alpha. These results indicate a non-hypoxic pathway mediating cytokine-dependent regulation of HIF-1alpha in a ROS-sensitive mechanism.

1. Pyrimidylpiperazine (Y-40138), a synthetic derivative of N-[1-(4-([4-(pyrimidin-2-yl)piperazin-1-yl]methyl)phenyl)cyclopropyl] acetamide, is a novel dual regulator of pro- and anti-inflammatory cytokines in vivo. The aim of the present study was to determine the signal transduction mechanisms implicated in vitro. 2. In alveolar epithelial cells, pre-treatment (30 min) with Y-40138 reduced LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha, an effect paralleled by up-regulating an anti-inflammatory counter-loop mediated through IL-10. 3. This differential regulation of pro- and anti-inflammatory signals was accompanied by an inhibition of the nuclear localization of selective NF-kappa B subunits, particularly NF-kappa B(1) (p50), RelA (p65), the major transactivating member of the Rel family, RelB (p68) and c-Rel (p75). In addition, Y-40138 blockaded, in a dose-dependent manner, the LPS-induced nuclear activation of NF-kappa B. 4. Analysis of the upstream pathway involved in Y-40138-dependent retardation of LPS-induced NF-kappa B translocation/activation revealed the involvement of an I kappa B-alpha sensitive pathway. Pre-treatment with Y-40138 ameliorated LPS-induced degradation of I kappa B-alpha in the cytosolic compartment and retarded its phosphorylation, suggesting the involvement of an upstream kinase. 5. Recombinant IL-10 (0 -- 10 ng ml(-1)) blockaded, in a dose-dependent manner, LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha. Furthermore, rhIL-10 reduced the DNA binding activity of NF-kappa B. Immunoneutralization of endogenous IL-10 by a polyclonal alpha IL-10 (5 microg ml(-1)) reversed the inhibitory effect of Y-40138 on pro-inflammatory cytokines and partially restored the DNA binding activity of NF-kappa B. 6. These results indicate that Y-40138 mediated dual immunoregulation of pro- and anti-inflammatory cytokines is IL-10 sensitive and mediated through the I kappa B-alpha/NF-kappa B signal transduction pathway.

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. 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. Reporter gene assays showed that no activation of the -ENaC (epithelial Na+ channel -subunit) promoter was discernible 24 h after PO2 was raised but increased transcriptional activity was seen at 48 h. 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. The increase in GNa thus coincides with activation of the -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 signalling mechanisms in oxidative stress mediated by cytokines in the perinatal alveolar epithelium are not well known. In an in vitro model of fetal alveolar type II epithelial cells, we investigated the profile of cytokines in response to ascending Deltap O(2)regimen (oxyexcitation). The peak of TNF-alpha (4 h) preceded IL-1beta and IL-6 (6-9 h), indicating a positive feedback autocrine loop confirmed by exogenous rmTNF-alpha. Reactive oxygen species (ROS) induced a dose-dependent release of cytokines, an effect specifically obliterated by selective antioxidants of the hydroxyl radical (*OH) and superoxide anion (O(2)-). Actinomycin and cycloheximide blocked the induced production of cytokines, implicating transcriptional and translational control. Whilst the dismutating enzymes superoxide dismutase (SOD) and catalase were ineffective in reducing ROS-induced cytokines, MnP, a cell-permeating SOD mimetic, abrogated xanthine/xanthine oxidase-dependent cytokine release. Desferrioxamine mesylate, which inhibits the iron-catalysed generation of *OH via the Fenton reaction, exhibited a mild effect on the release of cytokines. Dynamic variation in alveolar p O(2)constitutes a potential signalling mechanism within the perinatal lung allowing upregulation of cytokines in an ROS-dependent manner., (Copyright 2001 Academic Press.)

A self-referencing Cl--selective microelectrode (Cl- SrE) was developed and used to detect changes in the direction and magnitude of the Cl- flux (J(Cl)) from the apical region of cultured foetal distal lung epithelial cells (FDLEs) as a function of external Cl- concentration ([Cl-]e) and in response to pharmacological challenges. The technique, which is similar to that developed for other ion-selective microelectrodes, centres on the oscillation of a Cl--selective microelectrode between known points, micrometres apart, orthogonal to the plasma membrane. Application of the Fick principle to the differential voltage obtained per excursion amplitude (the referenced signal) yields the Cl- flux (pmol x cm(-2) x s(-1)). A Cl- effusion gradient was used to confirm that empirical measurements of J(Cl) using the Cl- SrE were statistically similar to predicted flux values calculated from the fall in [Cl-] with distance from the tip of the effusion source. Apical J(Cl) was then measured as a function of [Cl-]e from polarised FDLE cultures grown on permeable supports. At [Cl-]e<50 mmol x l(-1), an apical-to-basolateral (inward) flux, maximal at 400 pmol x cm(-2 ) x s(-1), was observed; this reverted to a continuous basolateral-to-apical (outward) flux of 203 pmol x cm(-2 ) x s(-1) at [Cl]e>100 mmol x l(-1). At [Cl-]e>100 mmol x l(-1), isoproterenol (basolaterally applied, 10 micromol x l(-1)) activated a Cl- influx of 561 pmol x cm(-2 ) x s(-1), whereas UTP (apically applied, 100 micromol x l(-1)) stimulated a Cl- efflux of 300 pmol x cm(-2) x s(-1). In all cases, 50-70 % of J(Cl) was abolished by Cl- channel blockade using 10 micromol x l(-1) diphenylamine-2-carboxylic acid (DPC) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that the Cl- SrE resolves a Cl- gradient in the microenvironment of the apical region of lung epithelia that varies in both direction and magnitude as a function of external [Cl-]e and in response to Cl- channel blockade and to beta2 adrenoreceptor and P2Y receptor agonists.

The O(2) and redox-sensitive transcription factors hypoxia inducible factor-1alpha (HIF-1alpha) and nuclear factor-kappaB (NF-kappaB) are differentially regulated in the alveolar epithelium over fetal to neonatal oxygen tensions. We have used fetal alveolar type II epithelial cells to monitor their regulation in association with redox responsiveness to antioxidant pretreatment in vitro. N-Acetyl-l-cysteine, a glutathione (GSH) precursor and a potent scavenger of reactive oxygen species, induced HIF-1alpha and ameliorated NF-kappaB nuclear abundance and DNA binding activity, respectively, in a dose-dependent manner. Analysis of variations in glutathione homeostasis at ascending DeltapO(2) regimen with N-acetyl-(L)-cysteine reveals increased GSH at the expense of the oxidized form of glutathione (GSSG), thereby shifting GSH/GSSG into reduction equilibrium. Pyrrolidine dithiocarbamate (PDTC), which exerts both antioxidant and pro-oxidant effects, provoked a substantial increase in HIF-1alpha nuclear abundance, with no apparent effect on its activation. PDTC reduced NF-kappaB nuclear abundance and its inhibitory effects on binding activity are dose-dependent. Assessment of glutathione homeostasis with PDTC shows increasing levels of GSSG at the expense of GSH, lowering GSH/GSSG in favor of an oxidative equilibrium. Our results indicate the hypoxic activation of HIF-1alpha and the hyperoxic induction of NF-kappaB in the fetal epithelium is redox-sensitive and, thus, tightly regulated by the GSH/GSSG equilibrium. This highlights glutathione as a key regulatory component for determining genetic responsiveness to oxidant/antioxidant imbalance in normal lung development and pathophysiological conditions.

To test the genetic capacity of the perinatal lung to respond to O(2) shifts that coincide with the first respiratory movements, rat fetal alveolar type II (fATII) epithelial cells were cultured at fetal distal lung PO(2) (23 Torr) and then exposed to postnatal (23 --> 76 Torr; mild hyperoxic shift), moderate (23 --> 152 Torr; moderate hyperoxic shift), or severe (23 --> 722 Torr; severe hyperoxic shift) oxygenation. Nuclear abundance and consensus binding characteristics of hypoxia-inducible factor (HIF)-1alpha and nuclear factor (NF)-kappaB (Rel A/p65) plus glutathione biosynthetic capacity were determined. Maximal HIF-1alpha activation at 23 Torr was sustained over the postnatal shift in (Delta) PO(2) and was elevated in vivo throughout late gestation. NF-kappaB was activated by the acute postnatal DeltaPO(2) in fATII cells, becoming maximal with moderate and severe oxygenation in vitro and within 6 h of birth in vivo, declining thereafter. fATII cell and whole lung glutathione and GSH-to-GSSG ratio increased fourfold with a postnatal DeltaPO(2) and were matched by threefold activity increases in gamma-glutamylcysteine synthetase and glutathione synthase. GSH concentration depletion by L-buthionine-(S, R)-sulfoximine abrogated both HIF-1alpha and NF-kappaB activation, with HIF-1alpha showing a heightened sensitivity to GSH concentration. We conclude that O(2)-linked genetic regulation in perinatal lung epithelium is responsive to developmental changes in glutathione biosynthetic capacity.

A self-referencing, polarographic, oxygen-selective microelectrode was developed for measuring oxygen fluxes from single cells. This technique is based on the translational movement of the microelectrode at a known frequency through an oxygen gradient, between known points. The differential current of the electrode was converted into a directional measurement of flux using the Fick equation. Operational characteristics of the technique were determined using artificial gradients. Calculated oxygen flux values matched theoretical values derived from static measurements. A test preparation, an isolated neuron, yielded an oxygen flux of 11.46+/-1.43 pmol cm-2 s-1 (mean +/- s.e.m.), a value in agreement with those available in the literature for single cells. Microinjection of metabolic substrates or a metabolic uncoupler increased oxygen flux, whereas microinjection of KCN decreased oxygen flux. In the filamentous alga Spirogyra greveilina, the probe could easily differentiate a 16.6% difference in oxygen flux with respect to the position of the spiral chloroplast (13.3+/-0.4 pmol cm-2 s-1 at the chloroplast and 11.4+/-0.4 pmol cm-2 s-1 between chloroplasts), despite the fact that these positions averaged only 10.6+/-1.8 microm apart (means +/- s.e.m.). A light response experiment showed real-time changes in measured oxygen flux correlated with changes in lighting. Taken together, these results show that the self-referencing oxygen microelectrode technique can be used to detect local oxygen fluxes with a high level of sensitivity and spatial resolution in real time. The oxygen fluxes detected reliably correlated with the metabolic state of the cell.