Cation currents in human airway epithelial cells induced by infection with influenza A virus.

Influenza A viruses cause lung disease via an incompletely understood mechanism that involves the accumulation of liquid within the lungs. The accumulation of lung liquid is normally prevented by epithelial Na(+) absorption, a transport process regulated via several pathways including phosphoinositide-3-kinase (PI3K). Since the influenza A virus encodes a non-structural protein (NS1) that can activate this kinase, we now explore the effects of NS1 upon the biophysical properties of human airway epithelial cells. Transient expression of NS1 depolarized electrically isolated cells maintained in glucocorticoid-free medium by activating a cation conductance identical to the glucocorticoid-induced conductance seen in single cells. This response involved PI3K-independent and PI3K-dependent mechanisms. Infecting glucocorticoid-deprived cells with influenza A virus disrupted the normal electrical coupling between neighbouring cells, but also activated a conductance identical to that induced by NS1. This response to virus infection was only partially dependent upon NS1-mediated activation of PI3K. The presence of NS1 allows influenza A to modify the biophysical properties of infected cells by activating a Na(+)-permeable conductance. Whilst the activation of Na(+)-permeable channels may be expected to increase the rate of Na(+) absorption and thus reduce the volume of liquid in the lung, liquid does normally accumulate in influenza A-infected lungs. The overall effect of influenza A on lung liquid volume may therefore reflect a balance between the activation and inhibition of Na(+)-permeable channels.

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.

Adenosine-evoked Na+ transport in human airway epithelial cells.

BACKGROUND AND PURPOSE: Absorptive epithelia express apical receptors that allow nucleotides to inhibit Na(+) transport but ATP unexpectedly stimulated this process in an absorptive cell line derived from human bronchiolar epithelium (H441 cells) whilst UTP consistently caused inhibition. We have therefore examined the pharmacological basis of this anomalous effect of ATP. EXPERIMENTAL APPROACH: H441 cells were grown on membranes and the short circuit current (I(SC)) measured in Ussing chambers. In some experiments, [Ca(2+)](i) was measured fluorimetrically using Fura -2. mRNAs for adenosine receptors were determined by the polymerase chain reaction (PCR). KEY RESULTS: Cross desensitization experiments showed that the inhibitory response to UTP was abolished by prior exposure to ATP whilst the stimulatory response to ATP persisted in UTP-pre-stimulated cells. Apical adenosine evoked an increase in I(SC) and this response resembled the stimulatory component of the response to ATP, and could be mimicked by adenosine receptor agonists. Pre-stimulation with adenosine abolished the stimulatory component of the response to ATP. mRNA encoding A(1), A(2A) and A(2B) receptor subtypes, but not the A(3) subtype, was detected in H441 cells and adenosine receptor antagonists could abolish the ATP-evoked stimulation of Na(+) absorption. CONCLUSIONS AND IMPLICATIONS: The ATP-induced stimulation of Na(+) absorption seems to be mediated via A(2A/B) receptors activated by adenosine produced from the extracellular hydrolysis of ATP. The present data thus provide the first description of adenosine-evoked Na(+) transport in airway epithelial cells and reveal a previously undocumented aspect of the control of this physiologically important ion transport process.

Perinatal PTX-sensitive G-protein expression and regulation of conductive 22Na+ transport in lung apical membrane vesicles.

Using apical membrane vesicles (AMV) prepared from mature foetal and early neonatal guinea pig lung we show that pertussis toxin (PTX)-sensitive G-protein regulation of conductive 22Na+ uptake undergoes rapid changes following birth. Thus, G-protein activation by intravesicular incorporation of 100 microM GTPgammaS into vesicles resuspended in NaCl, which in late gestation stimulated uptake, consistently induced inhibition of conductive Na+ uptake into AMV prepared from neonatal lung at 4 days of age (N4) (52+/-9%, n=8, P<0.05). This response was not significantly different in the presence of the relatively impermeant anion isethionate (Ise-) (69+/-9%, n=7, P<0.05). Changes in the regulation of uptake were already detectable on the day of birth (N0) in AMV resuspended in NaCl, with GTPgammaS inducing both stimulatory and inhibitory responses. These data indicate that the processes by which 22Na+ uptake into AMV is regulated by G-proteins undergoes a change at birth and by 4 days of age, G-protein regulation of uptake occurs predominantly via modulation of co-localised Na+ channels. Intravesicular incorporation of GDPbetaS or pre-treatment with PTX did not significantly alter conductive 22Na+ uptake in the presence of NaCl or NaIse suggesting that constitutively active G-proteins are not involved in this process. Pre-treatment of AMV with PTX prevented the inhibition of conductive 22Na+ uptake by GTPgammaS (105+/-16% n=7) indicating that a PTX-sensitive G-protein mediates the inhibition of channels in neonatal AMV. Western blotting demonstrated enrichment of Gialpha1, Gialpha2, Gialpha3 and Goalpha in the apical membrane preparations. We also show that there is a significant rise in the levels of Gialpha3 during the early neonatal period providing a potential candidate for the G-protein mediated changes in regulation of conductive 22Na+ uptake in neonatal AMV.

Conductive cation transport in apical membrane vesicles prepared from fetal lung.

In order to characterise the apically-located conductive cation pathway of the type II pneumocyte, apical plasma membranes were prepared from mature fetal guinea pig lung. The protocol yielded purified apical membranes that enriched 19-fold with the brush border enzyme marker alkaline phosphatase; there was no significant contamination with other cellular membranes. A technique for imposing an outwardly-directed electrochemical Na+ gradient was used to amplify conductive 22Na+ uptake into vesicles. Uptake of 22Na+ was time-dependent, proportional to the magnitude of the Na+ gradient, specific and sensitive to the amiloride analogues phenamil and EIPA (apparent minimum Ki values of 50 nM and 10 microM, respectively, with maximum uptake inhibition of 42% and 39% at 100 microM). Uptake experiments in which the outwardly-directed Na+ gradient was replaced by outwardly-directed gradients of small monovalent cations and molecular cations were performed. The Na+/K+ permeability ratio was 1.2:1, and over the extended range of small monovalent cations, a permeability sequence of Na+ > K+ > Li+ > Rb+ > Cs+ was observed, indicating the presence of fixed negative charge in or spatially close to the pore. The molecular cation permeability sequence of NH4+ > methylamine+ > dimethylamine+ > choline+ > N-methyl-D-glucamine+ > tetraethylammonium+ > tetramethylammonium+, after transformation, gives an estimate of 8 A for the conducting pore diameter. These data are consistent with the presence in the apical membrane of fetal type II pneumocytes of a cation specific channel with low Na+ selectivity and amiloride sensitivity.

Conductive Na+ transport in fetal lung alveolar apical membrane vesicles is regulated by fatty acids and G proteins.

We have characterised G protein and fatty acid regulation of the Na+ conductance in purified apical membrane vesicles prepared from late gestation fetal guinea-pig lung. Addition of 100 microM GTP gamma S or beta gamma-methylene-GTP, irreversible G protein activators, stimulated conductive 22Na+ uptake (ratio of experimental to control 1.35 +/- 0.02 and 1.34 +/- 0.05, respectively). Conversely, the addition of GDP beta S, an irreversible G protein inhibitor, reduced conductive 22Na+ uptake from 1.00 (control) to 0.79 +/- 0.04. A range of saturated (myristic, palmitic, stearic), monounsaturated (elaidic, oleic) and polyunsaturated (linoleic, arachidonic) fatty acids all stimulated conductive 22Na+ uptake, by between 1.18 +/- 0.05 to 1.56 +/- 0.13 over the control. Both arachidonic acid and GTP gamma S-dependent stimulation were abolished in the presence of 10 microM amiloride. The non-metabolisable analogue of arachidonic acid, eicosa-5,8,11,14-tetraynoic acid also stimulated conductive 22Na+ uptake. Furthermore, addition of indomethacin and nordihydroguairetic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonate metabolism respectively, did not affect the arachidonic acid stimulation suggesting a direct effect of fatty acid upon the Na+ channel Since mepacrine (50 microM), a phospholipase A2 inhibitor, did not affect the GTP gamma S-stimulated conductive 22Na+ uptake, and inhibition of G protein turnover by GDP beta S did not attenuate the arachidonic acid response we conclude that these two regulatory pathways modulate alveolar Na+ transport directly and independently of each other.

Transport of sodium into apical membrane vesicles prepared from fetal sheep alveolar type II cells.

A method is described for isolating apical plasma membrane vesicles from fetal alveolar type II cells. The procedure yields purified apical membranes which are enriched 24-fold with the brush-border enzyme marker, alkaline phosphatase. Contamination of this fraction by basolateral membranes and organelles is minimal. Evidence for transport of Na+ into an intravesicular space is demonstrated by: (1) time-dependent uptake of Na+ with release of accumulated Na+ by treatment with detergent; (2) a linear inverse correlation between Na+ uptake and medium osmolarity. In addition, Na+ uptake is shown to be anion dependent (SCN- greater than Cl- greater than gluconate-) and sensitive to amiloride inhibition at a concentration of 1 mM.

Differential regulation of Na+ and Cl- conductances by PTX-sensitive G proteins in fetal lung apical membrane vesicles.

In apical membrane vesicles (AMV) prepared from late gestation fetal guinea pig lung we show that conductive 22Na+ uptake is modulated by at least two pathways involving pertussis toxin (PTX)-sensitive G proteins. Intravesicular incorporation of 100 microM GTPgammaS into vesicles resuspended in NaCl caused a significant stimulation (P<0. 05) of conductive Na+ uptake in AMV to 150+/-10% (n=10) of control, whereas GDPbetaS reduced uptake to 65+/-9% (n=4) of control. This contrasting response to GTPgammaS and GDPbetaS is characteristic of a G protein mediated pathway. GTPgammaS induced a significantly smaller stimulation, 125+/-8% (n=5) of control, in the presence of the relatively impermeant anion isethionate (Ise-). Taken together, these data indicate modulation of both Na+ and Cl- channels in the apical membrane by co-localised G protein(s). Treatment with PTX stimulated conductive 22Na+ uptake to 171+/-20% (n=13) of control in AMV resuspended in NaCl, but did not have a significant effect, 94+/-19% of control, in the presence of NaIse indicating the existence of tonic activation of Cl- channels in these AMV under resting conditions. As the combined effects of PTX and GTPgammaS diminished uptake, we propose that the G protein(s) responsible for Na+ channel activation in response to GTPgammaS is PTX-sensitive and that additional PTX-insensitive G proteins might also modulate 22Na+ uptake in these AMV. The presence of Gialpha1, Gialpha2, Gialpha3 and Goalpha in this apical membrane preparation was confirmed by PTX catalysed [32P]ADP-dependent ribosylation and Western blotting. Incubation of AMV with 200 microM DTT caused an inhibition of conductive Na+ uptake in AMV resuspended in NaCl or NaIse to 66+/-8% (n=11) and 64+/-8% (n=6) of control respectively. Pre-treatment with DTT did not affect the ability of GTPgammaS to stimulate conductive Na+ uptake suggesting that the regulation of 22Na+ uptake in late gestation guinea pig fetal lung AMV is unlikely to involve an associated regulatory protein.

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, I(SC)) was monitored and effects of nucleotides upon I(SC) were studied. Mucosal UTP (100 microM) evoked a transient rise in I(SC) that was followed by a sustained fall below basal I(SC) maintained for 30 min. Mucosal ATP (100 microM) also stimulated a transient rise in I(SC) but in contrast to UTP did not inhibit basal I(SC). Submucosal UTP and ATP both transiently increased I(SC). 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 I(SC). The UTP-induced rise was reduced by pretreatment with either bumetanide (100 microM), diphenylamin-2-carboxylic acid (DPC, 1 mM), or Cl(-) and HCO(3)(-)-free solution whilst the fall was abolished by amiloride pretreatment. Thapsigargin (0.3 microM) abolished the UTP-induced increase in I(SC) but not the subsequent decrease. Staurosporine (0.1 microM) inhibited basal I(SC) and blocked UTP-induced inhibition of I(SC). 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 P2Y(2) receptors and an increase in [Ca(2+)](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.

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

1 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). 2 Apical ATP (100 microM) 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. 3 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. 4 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. 5 Basolateral isoprenaline (10 microM) 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 beta-adrenoceptors thus allow their respective agonists to exert mutually opposing effects on ISC.

Culture substrate-specific expression of P2Y2 receptors in distal lung epithelial cells isolated from foetal rats.

ATP and UTP did not evoke [Ca2+]i signals in rat foetal lung epithelial cells grown on glass but elicited clear responses in cells grown into functionally polarised epithelia on permeable supports. Moreover, P2Y2 receptor mRNA could not be detected in cells on glass by the polymerase chain reaction but this mRNA species was clearly expressed by polarised cells. P2Y2 receptor expression thus appears to be a feature of the polarised phenotype.

Dibutyryl cAMP induces a gestation-dependent absorption of fetal lung liquid.

The maturation of the adenosine 3',5'-cyclic monophosphate-(cAMP) dependent pathway controlling fetal lung liquid secretion was examined in experiments in which the lungs of chronically catheterized fetal lambs (123-141 days gestational age) were exposed to dibutyryl cAMP (DBcAMP, 10(-4) M). The effect of DBcAMP was markedly gestation dependent, with the greatest effect observed in the most mature fetuses. In immature fetuses (less than 130 days, mean age 125 days) DBcAMP caused slowing of secretion, with maximal effect at 5 h. With increasing maturity the effect of DBcAMP was more pronounced and occurred earlier so that in mature fetuses (mean age 140 days) lung liquid absorption took place, with maximal effect at 2 h. Changes in lung liquid volume flow induced by DBcAMP could be blocked by addition of 10(-4) M amiloride to lung liquid. It is concluded that 1) DBcAMP induces a change in lung liquid secretion that, like epinephrine, is mediated via an increase in Na+ permeability of the apical membrane of the lung epithelium and 2) the rate-limiting step in the maturation of this process must lie beyond the generation of intracellular cAMP.

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Expression of intermediate-conductance, Ca2+-activated K+ channel (KCNN4) in H441 human distal airway epithelial cells.

Electrophysiological studies of H441 human distal airway epithelial cells showed that thapsigargin caused a Ca(2+)-dependent increase in membrane conductance (G(Tot)) and hyperpolarization of membrane potential (V(m)). These effects reflected a rapid rise in cellular K(+) conductance (G(K)) and a slow fall in amiloride-sensitive Na(+) conductance (G(Na)). The increase in G(Tot) was antagonized by Ba(2+), a nonselective K(+) channel blocker, and abolished by clotrimazole, a KCNN4 inhibitor, but unaffected by other selective K(+) channel blockers. Moreover, 1-ethyl-2-benzimidazolinone (1-EBIO), which is known to activate KCNN4, increased G(K) with no effect on G(Na). RT-PCR-based analyses confirmed expression of mRNA encoding KCNN4 and suggested that two related K(+) channels (KCNN1 and KCNMA1) were absent. Subsequent studies showed that 1-EBIO stimulates Na(+) transport in polarized monolayers without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)), suggesting that the activity of KCNN4 might influence the rate of Na(+) absorption by contributing to G(K). Transient expression of KCNN4 cloned from H441 cells conferred a Ca(2+)- and 1-EBIO-sensitive K(+) conductance on Chinese hamster ovary cells, but this channel was inactive when [Ca(2+)](i) was <0.2 microM. Subsequent studies of amiloride-treated H441 cells showed that clotrimazole had no effect on V(m) despite clear depolarizations in response to increased extracellular K(+) concentration ([K(+)](o)). These findings thus indicate that KCNN4 does not contribute to V(m) in unstimulated cells. The present data thus establish that H441 cells express KCNN4 and highlight the importance of G(K) to the control of Na(+) absorption, but, because KCNN4 is quiescent in resting cells, this channel cannot contribute to resting G(K) or influence basal Na(+) absorption.

Ion transport and water flow in the mammalian lung.

The coupling of bulk water flow to active ion transport has been described in various epithelia; evidence presented here suggests that this is also a feature of the mammalian lung. Measurements of the ionic composition of lung liquid and its rate of formation in the fetal lamb in vivo have made it possible to estimate the net flux of each ion and, with water tracer measurements of ion one-way fluxes, to calculate flux ratios. When these are compared with the ratios predicted by the Ussing flux ratio equation it is clear that the secretion of lung liquid is linked to active transport of Cl- from plasma; sodium moves passively. In addition there is an apparent uphill transfer of HCO2- out of lung liquid. In an in vitro preparation of adult canine trachea Cl- is actively transported towards the lumen and is associated with a small net flux of Na+ in the opposite direction. Addition of acetylcholine increases the net Cl- flux towards the lumen but reverses the orientation of the net Na+ flux. Changes such as these may be important determinants of bulk liquid flow in vivo as well as in vitro.

Sodium and chloride transport by the tracheal epithelium of fetal, new-born and adult sheep.

In vitro measurements were made of Na+ and Cl- isotopic fluxes across the tracheal epithelium of mature fetal lambs (130-143 days gestation), new-born lambs (up to 41 days of age) and adult sheep under conditions of continuous short circuiting. The effects of a variety of drugs were examined, but only in the case of amiloride and isoprenaline were observations made in all three groups. Experiments designed to elucidate the mechanism of basal Cl- secretion were performed in adult trachea only. Under resting conditions the net flux of Na+ from lumen to submucosa exceeds that of Cl- in the reverse direction in fetal and adult trachea. In the new-born the two fluxes are more or less equivalent in magnitude. In none of the three groups is the sum of ion fluxes significantly different from the short-circuit current (Isc). Removal of Na+ from, or addition of furosemide (10(-3) M) to, the solution bathing the submucosal surface of adult trachea has the effect of reducing Isc by an amount which approximates to the Cl- current (29%). At a concentration of 10(-4) M on the submucosal side of adult trachea, ouabain causes potential difference and Isc to fall to zero within 70 min of addition to the bathing solution. Nevertheless, there remains a significant net Na+ flux from submucosa to lumen. The addition of isoprenaline (10(-4) M) to the medium bathing the submucosal surface of both fetal and adult trachea causes an increase in the one-way flux of Cl- from submucosa to lumen with consequent increase in net Cl- flux towards the lumen. (The Na+ fluxes are unchanged.) However, in the adult the Cl- secretory response to isoprenaline is very much less and is not accompanied by an increase in electrical conductance. As judged by the change in Isc, all the post-natal fall in beta-agonist responsiveness takes place within the 3 week period following birth. Whereas, in the fetus, the effect of luminally applied amiloride on the Na+ fluxes is negligible, in the adult the one-way flux of Na+ from lumen to submucosa is reduced by 35% with a consequent 60% fall in net Na+ flux towards the submucosa.(ABSTRACT TRUNCATED AT 400 WORDS)

G protein regulation of alveolar ion channels: implications for lung fluid transport.

Vectorial ion flow across the alveolar epithelium provides the driving force for lung fluid secretion in the prenatal lung and for fluid reabsorption at birth and thereafter into adult life. Fluid secretion is dependent upon 'active' accumulation of Cl- into the lumen of the developing lung and any factor which interupts the production of this liquid template results in life-threatening abnormalities in lung growth. The direction of fluid flow is reversed at birth to bring about the reabsorption of the lung fluid so that gaseous exchange can be initated successfully in the neonate. This functional switch at birth involves active Na+ reabsorption; failure of this mechanism to activate adequately contributes to respiratory distress in the newborn. Although beta 2-adrenoceptor-cAMP-protein kinase activation provides a basic model for the switch, it is clear that local regulation of many of the components in the switching cascade are important to the overall efficiency with which it is achieved. Co-localized with apical Cl- and Na+ channels are pertussis toxin-sensitive G proteins, which exert their regulatory effects principally by direct protein-channel interactions. The modulation of channels by local G proteins is finely tuned by negative feedback mechanisms, which may include a novel double-bond specific fatty acid regulation of G protein turnover and the paracrine effects of locally produced eicosanoids. An understanding of how these overlapping pathways integrate to produce the smooth and ordered transition from Cl- secretion to Na+ absorption will allow the design of rational treatments for conditions that are characterized by disfunctional lung fluid homeostasis.

The role of catecholamines in lung liquid absorption at birth.

We have examined the effect on lung liquid secretion of catecholamines infused in chronically catheterized fetal lambs in utero. Isoproterenol and epinephrine inhibited secretion, an effect which increased with gestation and, in fetuses near delivery, caused absorption of lung liquid. In 7 out of 8 experiments nor-epinephrine had no effect on secretion. This pattern of response and the fact that the inhibitory effect could be blocked by propranolol indicate a mode of action involving beta-adrenergic receptors.