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Journal of Physiology (2002), 544.2, pp. 567-577
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.022459
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ABSTRACT |
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Isolated rat fetal distal lung epithelial (FDLE) cells were cultured (~48 h) on permeable supports in medium devoid of hormones and growth factors whilst PO2 was maintained at the level found in either the fetal (23 mmHg) or the postnatal (100 mmHg) alveolar regions. The cells became incorporated into epithelial layers that generated a basal short-circuit current (ISC) attributable to spontaneous Na+ absorption. Cells at neonatal PO2 generated larger currents than did cells at fetal PO2, indicating that this Na+ transport process is oxygen sensitive. Irrespective of PO2, isoprenaline failed to elicit a discernible change in ISC, demonstrating that-adrenoceptor agonists do not stimulate Na+ transport under these conditions. However, isoprenaline did elicit cAMP accumulation in these cells, indicating that functionally coupled
(Received 19 April 2002; accepted after revision 25 July 2002; first published online 23 August 2002)
Corresponding author S. M. Wilson: Lung Membrane Transport Group, Tayside Institute of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK. Email: s.m.wilson{at}dundee.ac.uk
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INTRODUCTION |
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During fetal life, fluid is continually secreted into the lumen of the developing lung (Olver & Strang, 1974) establishing a distending pressure that is crucial to proper lung morphogenesis (Harding & Hooper, 1996). However, this liquid must be removed from the potential airspaces to allow breathing at birth, and studies of fetal lambs have shown that this is due to adrenaline-evoked liquid absorption. In relatively immature fetuses adrenaline slows the rate at which fluid accumulates within the lung lumen, but the magnitude of this inhibitory response increases with gestational age until, in mature fetuses, the hormone evokes overt liquid absorption rather than simply slowing the rate at which liquid accumulates (Walters & Olver, 1978; Brown et al. 1983). Irrespective of developmental status, these effects are mediated by -adrenoceptors and are mimicked by a cell-permeant cAMP analogue (Walters & Olver, 1978; Olver et al. 1986; Walters et al. 1990). Moreover, adrenaline- and cAMP-evoked liquid absorption are both blocked by amiloride (Olver et al. 1986; Walters et al. 1990), a Na+ channel antagonist that also abolishes the naturally occurring absorption of lung liquid seen during labour and birth (O'Brodovich et al. 1990). As -adrenoceptor antagonists also inhibit this process (Brown et al. 1983), this body of work indicates that adrenaline-evoked Na+ transport underlies the absorption of lung liquid seen at birth. Subsequent studies of genetically modified mice have shown that this process is also abolished by introducing null mutations into both epithelial Na+ channel 
subunit (-ENaC) alleles (Hummler et al. 1996). Lung liquid absorption can thus be attributed to -adrenoceptor-mediated control over ENaC.
The mechanisms underlying this control have since been studied extensively using isolated rat fetal distal lung epithelial (FDLE) cells maintained in short-term culture (see review by Matalon & O'Brodovich, 1999). Such experiments confirmed that -adrenoceptors allow adrenaline to stimulate Na+ transport in fetal alveolar epithelia and showed that this response involves a rise in apical membrane Na+ conductance (GNa) (see for example Ito et al. 1997; Marunaka et al. 1999; Matalon & O'Brodovich, 1999). However, whilst these findings seem to accord with data from fetal lambs (Brown et al. 1983; Olver et al. 1986; Walters et al. 1990), more recent studies have revealed a significant problem with this system. This difficulty stems from the fact that almost all studies of FDLE cells were undertaken using cells cultured in an atmosphere of water-saturated room air containing 5 % CO2. The PO2 of this gas mixture is ~142 mmHg, which is greater than that found in either the fetal (~23 mmHg) or the neonatal alveolar region (~100 mmHg). The realisation that alveolar Na+ transport is intrinsically sensitive to PO2 (Barker & Gatzy, 1993; Pitkänen et al. 1996; Rafii et al. 1998) prompted experiments in which FDLE cells were maintained in an atmosphere in which PO2 was controlled (Haddad & Land, 2000; Ramminger et al. 2000; Baines et al. 2001; Haddad et al. 2001; Collett et al. 2002). These studies confirmed that isoprenaline stimulates Na+ transport when PO2 is high (Ramminger et al. 2000; Collett et al. 2002), but showed that this drug failed to elicit any response in cells maintained at the PO2 experienced in utero (Ramminger et al. 2000). Data from rat FDLE cells thus contrast with data from in vivo studies of the mature fetal lamb, where -adrenoceptor agonists can clearly stimulate lung liquid clearance (Walters & Olver, 1978; Olver et al. 1986). Rat FDLE cells thus fail to retain an important physiological feature of the fetal lung when maintained under the conditions used in earlier studies. To explore the possibility that modifying the culture regime might allow better retention of a fetal phenotype, we now explore the effects of exogenous hormones upon the sensitivity of FDLE cells to the -adrenoceptor agonist isoprenaline. These experiments show that thyroid and glucocorticoid are necessary for -adrenoceptor-mediated control over GNa. Some of these data have been presented to The Physiological Society (Ramminger et al. 2001, 2002; Olver et al. 2002 ).
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METHODS |
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Solutions and chemicals
Physiological salt solution contained (mM): NaCl 117, NaHCO3 25, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5 and D-glucose 11; pH 7.3-7.4 when bubbled with 5 % CO2. The sodium gluconate solution was prepared by iso-osmotically replacing Cl- in this standard solution with gluconate, whilst the K+-Cl- solution was prepared by replacing Na+ with K+. Both ionic substitutions were made in the potassium gluconate solution. The amount of calcium gluconate added to gluconate-containing solutions was raised to 11.5 mM in order to maintain Ca2+ activity despite gluconate's capacity to bind this cation. The Hepes-buffered solution contained (mM): NaCl 112, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, Hepes (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) 20, and D-glucose 11 (pH adjusted to 7.3-7.4 with NaOH). All other solutions were bicarbonate buffered and continually bubbled with 5 % CO2 to maintain the pH. The standard, minimally defined serum-free (MDSF) medium consisted of a mixture (1:1) of Dulbecco's Modified Eagle's Medium- Ham's F-12 nutrient mix containing bovine serum albumin (1.25 mg ml-1), L-glutamine (2 mM) and non-essential amino acids (0.1 %).
Isolation and culture of FDLE cells
Time-mated, pregnant rats were anaesthetised (3 % halothane) 72 h before the pregnancy's full term (22 days) and their fetuses delivered by Caesarean section and immediately decapitated. The anaesthetised animals were then killed without regaining consciousness. These procedures were carried out in accordance with the legislation currently in force in the UK and with the University of Dundee's animal welfare guidelines. Fetal lung tissue was collected into ice-cold Hanks' balanced salt solution, and chopped into pieces that were disaggregated using trypsin and collagenase. FDLE cells were isolated from the resultant digest by differential centrifugation/adhesion onto plastic (see Ramminger et al. 2000) and plated onto Transwell Col membranes. Initially, all cells were incubated (37 °C) in standard MDSF medium for ~24 h in a water-saturated atmosphere (PH2O = 47 mmHg) containing 5 % CO2 (PCO2 = 38 mmHg) and sufficient N2 to lower PO2 to the level found in either the fetal (23 mmHg) or postnatal alveolar regions (100 mmHg). Each culture was then washed gently and incubated in fresh medium for ~24 h before being used in experiments. During this second incubation period, cells were maintained in either standard MDSF medium, or in MDSF medium supplemented with tri-iodothyronine (T3, 10 nM) and/or dexamethasone (200 nM).
Transepithelial ion transport and apical membrane conductive properties
Cultured epithelia were mounted in Ussing chambers, bathed with physiological saline and initially maintained under open-circuit conditions whilst transepithelial voltage (Vt) was monitored. Once this parameter had stabilised (30-40 min), Vt was clamped to 0 mV and the current needed to maintain this potential difference (the short-circuit current, ISC) monitored and recorded to computer disk using a PowerLab interface and associated software (ADInstruments, Hastings, UK). The cited values of transepithelial resistance (Rt) were calculated by solving Ohm's Law using the values of ISC and Vt measured at the onset of each experiment. The conductive properties of the apical membrane were explored as described previously (Collett et al. 2002). Briefly, cultured epithelia were first bathed symmetrically with standard physiological saline. Three aliquots of saline were then withdrawn from both the apical and basolateral baths and each replaced successively with an equal volume of the potassium gluconate solution. In this way the external solution was diluted with potassium gluconate solution (8.1:91.9) to give a cytoplasm-like solution in which the concentrations of principal ions were (mM): Na+ 11.5, K+ 135.3, Cl- 10.3, gluconate 122.0. The basolateral membrane was then permeabilised by adding nystatin (75 µM) to the basolateral bath. To measure apical Na+ conductance, an inwardly directed Na+ gradient was imposed upon the cells by withdrawing a 5 ml aliquot of the cytoplasm-like solution from the apical bath and replacing it with 5 ml of a solution prepared by diluting (8.1:91.9) the standard salt solution with sodium gluconate solution. The concentration of Na+ in the apical bath was thus raised to 55 mM by iso-osmotically replacing K+, whilst the concentrations of other ions remained constant. Under these conditions the driving force for Na+ entry (VNa) is determined by the difference between Vt (0 mV) and the equilibrium potential for Na+ (ENa, 41.8 mV). GNa can thus be calculated using the expression GNa = Iamil/VNa, in which Iamil is the change in apical membrane current elicited by apical amiloride (10 µM). Our previous work has shown that isoprenaline has no acute effect upon GNa in permeabilised preparations, but that clear increases in GNa could be seen if cells are permeabilised after the isoprenaline-evoked increase in ISC has become fully established (Collett et al. 2002). We therefore designed the present experiments so that we could compare GNa in isoprenaline-stimulated cells with the equivalent values from age-matched control cells that had been isolated from the same litters and maintained in Ussing chambers for an essentially identical period of time. To measure apical Cl- conductance (GCl), an outwardly directed Cl- gradient was imposed upon the basolaterally permeabilised cells by replacing some of the gluconate in the basolateral bath with Cl- and so raising the Cl- concentration to 49 mM. Previous work has shown that cAMP-coupled agonists elicit rapid increases in apical membrane current under such conditions, and these responses can be attributed to increased activity of apical anion channels (Jiang et al. 1998; Collett et al. 2002). In the present study, although we consistently observed clear responses to isoprenaline, the magnitude of the resting, apical membrane Cl- current was variable. Basal currents recorded from cells maintained in MDSF medium at neonatal alveolar PO2 thus ranged from 1.2 to 8.7 µA cm-2. As Rt was ~400 
cm2, this variability indicates that an unexplained asymmetry of 0.5-4 mV persisted despite attempts to correct liquid junction potentials empirically (see Collett et al. 2002). The presence of this variable resting current substantially increased the variance in our pooled current records and so, to facilitate the presentation of these data, the resting current was subtracted from all records. Responses to isoprenaline are thus shown as increases in apical membrane current. In all electrophysiological experiments, a positive ISC is defined as the current carried by cations moving from the apical to the basolateral compartments whilst, in basolaterally permeabilised preparations, positive apical membrane current is defined as that carried by cations leaving the cytoplasm.
Na+ pump capacity
To measure basolateral Na+ pump capacity, cultured epithelia bathed with standard physiological solution were first exposed to apical amiloride (10 µM) to block the Na+ channels in this membrane, which was then permeabilised using nystatin (75 µM). This elicited a slowly developing rise in current that was attributable to the activity of the basolaterally located Na+ pump. The fall in current evoked by subsequently adding ouabain (1 mM) to the basolateral solution was then measured as an indicator of this pump's Na+ extrusion capacity. Previously published work (Ramminger et al. 2000) shows that 1 mM ouabain never entirely abolished the current recorded under these conditions, indicating that the method may underestimate the pump capacity. We are forced to accept this limitation, however, as higher concentrations of ouabain cause loss of epithelial integrity.
Cellular cAMP content
Cells grown to confluence on Transwell membranes were washed briefly with Hepes-buffered saline containing the phosphodiesterase inhibitor isomethylbutylxanthine (IBMX, 5 mM) and incubated in this solution for 10 min before experiments were initiated by adding isoprenaline (final concentration 10 µM) to the basolateral solution. Experiments were carried out on a heated surface in order to maintain the incubating solutions at 37 °C. After 2 min the cells were removed from the isoprenaline-containing solution and lysed with perchloric acid (final concentration 0.1 M) in order to extract cAMP. The acid extracts were recovered and neutralised with 60 mM Hepes containing 0.1 % Universal Indicator. Cellular proteins, which are characteristically insoluble in perchloric acid, were subsequently extracted using 0.1 M NaOH and assayed using Bradford reagent. The amount of cAMP present in each acid extract was determined by radioimmunoassay (Rakhit et al. 1998) and data are presented as picomoles of cAMP per microgram of cellular protein (pmol µg-1).
Experimental design and data analysis
Data are shown as means ± S.E.M. and values of n refer to the number of times a protocol was repeated using cells prepared from different litters. All experiments were undertaken using strictly paired protocols in which control and experimental cells were age matched and derived from the same litters; data from hormone-treated cells have thus been compared directly (Student's paired t test) with equivalent data from control cells that were maintained in standard MDSF medium, but otherwise treated identically.
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RESULTS |
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Effects of PO2 upon cells maintained in MDSF medium
The values of Rt and Vt derived from cells maintained at fetal PO2 in control medium (i.e. containing no hormones or growth factors) were lower than the equivalent values derived from cells maintained at postnatal PO2 (Table 1). Measurements made from these cells under short-circuit conditions showed that the cells maintained at elevated PO2 also generated a larger spontaneous ISC (postnatal PO2, 10.6 ± 0.4 µA cm-2; fetal PO2, 6.1 ± 0.4 µA cm-2; P < 0.001). As anticipated by earlier work (see for example Pitkänen et al. 1996; Matalon & O'Brodovich, 1999; Ramminger et al. 2000; Baines et al. 2001), apical amiloride caused a substantial fall in ISC at both O2 tensions. This occurred with no discernible latency and the current stabilised at its new value 20-30 s after adding the drug to the bath. Ambient PO2 did not affect the magnitude of this amiloride-resistant current, and so elevating PO2 selectively augments the amiloride-sensitive component of the basal ISC (fetal PO2, 3.7 ± 0.4 µA cm-2; postnatal PO2, 8.3 ± 0.6 µA cm-2; P < 0.001). This agrees well with earlier data from cells cultured in a commercially available supplemented medium (PC-1), which showed that this oxygen-evoked stimulation of Na+ transport was due to increases in Na+ pump capacity and GNa (Ramminger et al. 2000; Baines et al. 2001). Further experiments showed that such increases in Na+ pump capacity (fetal PO2, 3.7 ± 0.4 µA cm-2; postnatal PO2, 6.9 ± 0.7 µA cm-2; n = 22, P < 0.0001) and GNa (fetal PO2, 44.0 ± 8.4 µS cm-2; postnatal PO2, 104.0 ± 17.2 µS cm-2; n = 8, P < 0.001) also occurred under the present conditions.
Effects of isoprenaline upon cells maintained in MDSF medium
Irrespective of PO2, basolateral isoprenaline (10 µM) failed to elicit a discernible change in ISC in cells cultured in control medium (Fig. 1, Table 2). This contrasts with data from cells maintained in PC-1 medium, and other such supplemented media, where this drug clearly increases ISC when PO2 is high (see for example Matalon & O'Brodovich, 1999; Ramminger et al. 2000). Even under these conditions, however, cells do not respond to isoprenaline at fetal PO2 (Ramminger et al. 2000).
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Figure 1. Isoprenaline-evoked changes in short-circuit current (ISC) Isolated fetal distal lung epithelial (FDLE) cells were cultured for ~48 h at O2 tensions mimicking those found in either the postnatal (A) or fetal alveolar region (B). In both instances the control cells were maintained in standard, minimally defined serum-free (MDSF) medium, whilst hormone (Dex + T3)-treated cells were exposed to MDSF medium containing dexamethasone and T3 for the final 24 h of this period. ISC was then monitored under unstimulated conditions and during stimulation with basolateral isoprenaline (10 µM), which was delivered as indicated by the arrows (Iso). In each panel the continuous lines show the mean ISC recorded in the entire series of experiments (A, control: n = 7, transepithelial resistance, (Rt) = 619 ± 37 | ||
Effects of dexamethasone
The values of Rt and Vt derived from cells cultured in the presence of dexamethasone (200 nM, n = 9) for ~24 h did not differ significantly from values measured in age-matched control cells. Moreover, elevated PO2 caused increases in both Rt and Vm in the dexamethasone-treated cells that did not differ from those seen under control conditions (Table 1). Measurements made under short-circuit conditions showed that treatment with dexamethasone increased basal ISC by ~25 % at both fetal PO2 (control, 5.1 ± 0.6 µA cm-2; dexamethasone-treated, 6.0 ± 0.7 µA cm-2; P < 0.002) and at postnatal PO2 (control, 8.4 ± 0.7 µA cm-2; dexamethasone-treated, 9.8 ± 0.9 µA cm-2; P < 0.01). Further analysis of these data also showed that the oxygen-evoked stimulation of ISC seen in cells cultured in the control medium (see above) also occurred in cells maintained in the presence of dexamethasone (P < 0.005). However, inclusion of dexamethasone in the culture medium did not allow isoprenaline to evoke discernible increases in ISC at either fetal or postnatal PO2 (Table 2).
Effects of T3
The values of Rt and Vt derived from cells cultured in the presence of T3 (10 nM, n = 7) for ~24 h were comparable to control and displayed similar dependence upon PO2 (Table 1). Cells cultured in T3-containing medium had elevated basal ISC at both O2 tensions (fetal PO2: control, 5.1 ± 0.6 µA cm-2; T3-treated, 7.7 ± 0.9 µA cm-2; P < 0.05; postnatal PO2: control, 11.4 ± 0.9 µA cm-2; T3-treated, 14.6 ± 1.3 µA cm-2; P < 0.05) and analysis of these data showed clearly that ISC generated by these cells was sensitive to ambient PO2 (P < 0.01). However, culture in medium containing T3 did not allow isoprenaline to exert control over ISC at either fetal or postnatal PO2 (Table 2).
Effects of simultaneous exposure to dexamethasone and T3
Cells cultured in the presence of both dexamethasone and T3 became incorporated into electrically resistive monolayers and, although PO2 had no effect on Rt in these cells, it did cause a rise in Vt (Table 1). At both O2 tensions, the spontaneous ISC recorded from cells maintained in the presence of these hormones was greater than that recorded from control cells (fetal PO2: control ISC, 8.1 ± 0.5 µA cm-2; hormone-treated, 10.9 ± 1.3 µA cm-2; n = 20, P < 0.05; postnatal PO2: control ISC, 11.7 ± 0.7 µA cm-2; hormone-treated, 14.8 ± 1.5 µA cm-2; n = 22, P < 0.01) but these effects of PO2 did not differ from those seen in cells treated with the individual hormones. Analysis of these data also showed that increased PO2 caused a rise (P < 0.01) in spontaneous ISC in dexamethasone and T3-treated cells. However, the most important result to emerge from these experiments was that isoprenaline evoked clear increases in ISC in cells cultured in the presence of both hormones, although the response seen at fetal PO2 (Fig. 1A) was smaller than at postnatal PO2 (Fig. 1B, Table 2). In subsequent experiments, dexamethasone and T3-treated cells maintained at fetal (n = 3) or postnatal PO2 (n = 4) were exposed to apical amiloride for 2-3 min before being stimulated with basolateral isoprenaline (10 µM). Under these conditions isoprenaline had no discernible effect upon amiloride-treated cells, demonstrating that isoprenaline acts by increasing the amiloride-sensitive component of the basal ISC.
Isoprenaline-evoked changes in GNa
Cells were maintained at postnatal PO2 in control medium, or in medium supplemented with dexamethasone and T3, and ISC then recorded under unstimulated conditions and during exposure to isoprenaline (10 µM). Data from control cells confirmed that isoprenaline does not cause a discernible rise in ISC in the absence of hormones and growth factors (Fig. 2A). Thus, the current recorded after 40 min exposure to this drug (8.3 ± 0.7 µA cm-2) was essentially identical to that recorded from the unstimulated cells (Fig. 2B, 7.7 ± 0.2 µA cm-2). Once these recordings were completed, the cells were basolaterally permeabilised so that GNa could be measured (Collett et al. 2002); in cells that had been maintained in control medium, this analysis showed that GNa was essentially identical in unstimulated and isoprenaline-stimulated cells (Fig. 2C). In contrast, isoprenaline elicited a clear increase in ISC in cells cultured in the presence of dexamethasone and T3 (Fig. 2D); the current recorded after 40 min exposure to this drug (14.9 ± 0.4 µA cm-2) was thus greater (P < 0.05) than that measured in the corresponding unstimulated cells (Fig. 2E, 12.2 ± 0.9 µA cm-2). Subsequent measurements of GNa showed that the increase in ISC seen in isoprenaline-stimulated cells was matched by a corresponding rise in GNa (Fig. 2C).
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Figure 2. The records presented in A and B show ISC recorded from cultured epithelia that had been maintained in standard MDSF medium at postnatal PO2; the recordings were made both during stimulation with 10 µM basolateral isoprenaline (A, n = 5, Rt = 838 ± 105 | ||
This experimental protocol was also used to study cells cultured at fetal PO2. These experiments confirmed that isoprenaline failed to increase ISC in cells maintained in medium devoid of hormones and growth factors (unstimulated, 4.5 ± 0.7 µA cm-2; isoprenaline-stimulated, 4.8 ± 0.9 µA cm-2) and showed that isoprenaline has no effect upon GNa under these conditions (unstimulated, 30.1 ± 4.6 µS cm-2; isoprenaline-stimulated, 36.5 ± 4.0 µS cm-2). However, isoprenaline elicited a clear increase in ISC in dexamethasone and T3-treated cells (unstimulated, 4.3 ± 0.6 µA cm-2; isoprenaline-stimulated, 7.3 ± 1.0 µA cm-2; P < 0.001) and this response was associated with a clear rise in GNa (unstimulated, 41.6 ± 5.9 µS cm-2; isoprenaline-stimulated, 65.1 ± 11.0 µS cm-2; P < 0.02).
Isoprenaline-evoked cAMP formation
The data presented above suggest that dexamethasone and T3 are needed to allow isoprenaline to control GNa. We therefore explored the possibility that the insensitivity to isoprenaline seen in cells maintained under control conditions might reflect a lack of functionally coupled -adrenoceptors by measuring isoprenaline-evoked cAMP accumulation in control cells and in dexamethasone and T3-treated cells. Experiments undertaken at neonatal alveolar PO2 showed that the hormonal status of the cells had no significant effect upon basal cAMP content and established that isoprenaline could evoke cAMP accumulation in both groups of cells (Fig. 2F). There was no significant difference between the responses seen in control and hormone-treated cells (Fig. 2F). Studies of cells maintained at fetal PO2 (n = 9) showed that hormonal status also had no effect upon basal cAMP content under these conditions (control, 4.1 ± 1.5 pmol µg-1; dexamethasone and T3-treated, 6.1 ± 2.4 pmol µg-1) and demonstrated that isoprenaline evoked essentially identical increases in cAMP content in both groups of cells (control, 2.2 ± 0.8 pmol µg-1; dexamethasone and T3-treated, 2.0 ± 0.8 pmol µg-1). Although the mean cAMP levels measured in cells maintained at neonatal PO2 appeared greater than those measured in cells cultured at fetal PO2, this difference was not statistically significant.
Effects of IBMX
The measurements of cellular cAMP content were undertaken using cells treated (10 min) with 5 mM IBMX, a phosphodiesterase inhibitor that was included to inhibit cAMP hydrolysis, and showed that isoprenaline could elicit cAMP synthesis in control and hormone-treated cells (Fig. 2F). We therefore explored the possibility that IBMX might allow isoprenaline to modulate ISC in cells maintained under hormone- and growth-factor-free medium. Initially, we studied the effects of IBMX (5 mM) upon basal ISC by comparing (Student's paired t test) the current recorded immediately before adding IBMX to the apical and basolateral baths with that measured after 10 min exposure to this substance. IBMX evoked slowly developing increases in ISC in the hormone-treated cells maintained at either fetal (basal ISC, 3.3 ± 0.5 µA cm-2; IBMX-treated ISC, 5.6 ± 1.2 µA cm-2; P < 0.05, n = 4) or postnatal alveolar PO2 (basal ISC, 10.6 ± 1.8 µA cm-2, IBMX-treated ISC: 13.4 ± 1.7 µA cm-2; P < 0.01, n = 5). Qualitatively, this response was similar to that evoked by isoprenaline. However, IBMX had no effect upon cells cultured in hormone-free medium (fetal PO2: basal ISC, 3.7 ± 0.5 µA cm-2; IBMX-treated ISC, 3.7 ± 0.4 µA cm-2; n = 4; neonatal PO2: basal ISC, 8.8 ± 0.9 µA cm-2; IBMX-treated ISC, 9.1 ± 0.9 µA cm-2; n = 5). IBMX can thus evoke increased ISC, but this response, in common with the response to isoprenaline, is dependent upon the presence of dexamethasone and T3. After 10 min, the IBMX-treated cells were exposed to basolateral isoprenaline (10 µM). This caused a further increase in ISC in the hormone-treated cells (n = 4) maintained at either fetal (
ISC = 2.1 ± 0.4 µA cm-2, P < 0.02) or postnatal PO2 (ISC = 3.0 ± 1.1 µA cm-2, P < 0.01), but had no effect upon cells maintained in hormone-free medium (fetal PO2: ISC = 0.1 ± 0.2 µA cm-2; neonatal PO2: ISC = -2.1 ± 1.3 µA cm-2; n = 5). IBMX thus fails to allow isoprenaline to exert control over ISC in cells maintained under hormone- and growth-factor-free conditions.
Isoprenaline-evoked changes in GCl
Previous studies of FDLE cells maintained in supplemented media showed that, as well as controlling GNa, -adrenoceptor agonists rapidly increase GCl (Collett et al. 2002). We therefore explored the acute effects of isoprenaline upon the currents flowing across basolaterally permeabilised epithelia exposed to an outwardly directed Cl- gradient to investigate the possibility that dexamethasone and T3 may be involved in the regulation of this response. At neonatal PO2, isoprenaline evoked rapid, inward currents in cells maintained in control medium (Fig. 3A) or in medium supplemented with dexamethasone and T3 (Fig. 3B). Although the response seemed larger in hormone-treated cells (Fig. 3C), this apparent effect was not statistically significant. Isoprenaline also evoked inward current in cells maintained at fetal PO2 (Fig. 3D and E) and the cells' hormonal status had no significant effect upon this response (Fig. 3F). Further analysis of these data showed that ambient PO2 did not influence the isoprenaline-evoked rise in GCl in control cells (Fig. 3C and F), but that the response recorded from hormone-treated cells was larger (P < 0.02) at postnatal PO2 (Fig. 3C) than at fetal PO2 (Fig. 3F). However, the important point to emerge from these experiments is that at both O2 tensions, isoprenaline evoked clear increases in GCl in cells cultured in the absence of hormones and growth factors. This shows that cells maintained under these conditions have the ability to respond to isoprenaline, and so this drug's inability to increase ISC in cells maintained in medium devoid of hormones and growth factors must reflect a selective disruption of control over GNa.
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Figure 3. Isoprenaline-evoked apical membrane Cl- currents The currents flowing across basolaterally permeabilised cells exposed to an outwardly directed Cl- gradient were recorded during stimulation with isoprenaline (10 µM). Experiments were undertaken using cells incubated at postnatal (A, B, C) or fetal (D, E, F) alveolar PO2 either in standard MDSF medium (A, D; Control) or in MDSF medium supplemented with 200 nM dexamethasone and 10 nM T3 (B, E; Dex + T3). The traces show the mean apical membrane currents recorded under each of the four experimental conditions (fetal PO2: control, n = 6, Rt = 433 ± 89 | ||
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DISCUSSION |
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Properties of FDLE cells maintained in MDSF medium
Most previous studies of cultured FDLE cells have been undertaken using medium supplemented with hormones and growth factors; these were either added to the medium directly or were present in fetal bovine serum, which was used as a culture supplement. The present experiments therefore explored the effects of maintaining cells in medium devoid of such additives. These studies showed clearly that hormones and/or growth factors are not necessary to allow FDLE cells to become incorporated into electrically resistive epithelial sheets, a finding that confirms the results of an earlier investigation (O'Brodovich et al. 1992). Moreover, these cultured epithelia generated a spontaneous ISC due largely to the absorption of Na+ from the apical solution. The magnitude of this spontaneous current was greater when PO2 was elevated, and studies of permeabilised cells showed that this stimulation of ISC was due to increases in Na+ pump capacity and GNa. These data agree well with results from cells maintained in supplemented medium (Ramminger et al. 2000; Baines et al. 2001) and thus confirm that a rise in PO2 resembling that seen as the newborn infant takes its first breaths at birth is an effective stimulus for Na+ transport in FDLE cells. This rise in PO2 may thus provide a stimulus that is important to the functional maturation of the lung (Barker & Gatzy, 1993; Pitkänen et al. 1996; Rafii et al. 1998; Ramminger et al. 2000; Baines et al. 2001; Haddad et al. 2001).
Irrespective of PO2, isoprenaline failed to increase ISC in cells maintained in the standard MDSF medium, a finding that contrasts with data from fetal and adult alveolar epithelial cells maintained in supplemented media, in which this drug stimulates Na+ transport when PO2 is high (see for example Ito et al. 1997; Marunaka et al. 1999; Matalon & O'Brodovich, 1999; Ramminger et al. 2000; Collett et al. 2002). MDSF medium thus lacks a component/components needed for -adrenoceptor-mediated control of Na+ transport, and subsequent experiments were therefore directed towards defining culture conditions that might allow sensitivity to this -adrenoceptor agonist to be retained.
The effects of exogenous hormones
As thyroid and glucocorticoid hormones influence the development of the surfactant-secreting mechanism and the functional maturation of pulmonary ion transport processes (for example see Gross et al. 1984; Barker et al. 1988, 1990a; Renard et al. 1995; Tchepichev et al. 1995; Barquin et al. 1997; Grubb & Boucher, 1998; Norlin et al. 1999; Otulakowski et al. 1999), we first explored the effects of maintaining FDLE cells in medium supplemented with either T3 or dexamethasone. Data from unstimulated cells showed that these hormones increased basal ISC, indicating that they can stimulate Na+ transport. Despite these clear effects, isoprenaline failed to elicit a discernible response in cells treated with either hormone and so, in further experiments, we explored the effects of exposing cells to both hormones. This caused an increase in basal ISC that was similar to that seen when the hormones were administered individually, indicating that their effects upon basal ISC are not additive. However, cells maintained in the presence of both hormones displayed clear responses to isoprenaline at both fetal and adult alveolar PO2, demonstrating that simultaneous exposure to dexamethasone and T3 can restore sensitivity to this -adrenoceptor agonist. It is interesting, however, that at postnatal PO2 the ISC generated by unstimulated cells that had been treated with T3 alone were similar in magnitude to the currents recorded from isoprenaline-stimulated cells that had been cultured in the presence of dexamethasone and T3 (Table 2). This observation raises the possibility that T3 may increase ISC by activating the isoprenaline-sensitive component of the ISC and, if this model is accepted, then it is possible that the inclusion of dexamethasone may restore sensitivity to isoprenaline by effectively suppressing this stimulatory effect of T3. This model cannot, however, be formally established on the basis of the present data, but the possibility that these hormones may act in this way is worthy of further study.
Studies of cells cultured in the presence of dexamethasone and T3 clearly show that isoprenaline can evoke increased ion transport in cells maintained at either fetal or postnatal PO2. This contrasts with the results of earlier experiments in which cells were maintained in medium PC-1, which contains the hormones/growth factors normally found in serum. Under these conditions, isoprenaline increased Na+ transport at postnatal PO2 but not at fetal PO2 (Ramminger et al. 2000). We therefore believe that the present study is the first to demonstrate -adrenoceptor-mediated control of Na+ transport in cultured FDLE cells maintained at fetal PO2, and that the culture conditions used in the present study allow better retention of a fetal phenotype. It was clear, however, that the response to isoprenaline was larger at adult alveolar PO2, which suggests that as well as stimulating basal Na+ transport (Barker & Gatzy, 1993; Pitkänen et al. 1996; Rafii et al. 1998; Ramminger et al. 2000; Baines et al. 2001; Haddad et al. 2001), increased PO2 might augment -adrenoceptor-mediated liquid absorption. This could have important consequences, as studies in a number of species show that an appreciable volume of liquid remains in the lungs at birth and that absorption continues into the postnatal period (Bland et al. 1980; Brown et al. 1983; O'Brodovich et al. 1990). The rise in PO2 that occurs as the newborn infant takes its first breaths may thus play an important role in pulmonary physiology by augmenting the effects of circulating adrenaline as well as stabilising the Na+ absorbing phenotype.
Dexamethasone and T3 thus act synergistically to permit -adrenoceptor-mediated control of Na+ transport in FDLE cells and it is interesting, in this context, that the development of adrenaline-evoked fluid absorption in the fetal lamb is blocked by fetal thyroidectomy (Barker et al. 1988). Although this effect was reversed by infusion of T3 (Barker et al. 1990b), delivering T3 to immature fetuses did not cause precocious maturation of the response unless given in conjunction with a glucocorticoid (Barker et al. 1990a). The functional maturation of the lung in utero thus seems to be dependent upon simultaneous exposure to thyroid and glucocorticoid hormones (Gross et al. 1984; Barker et al. 1990a) and it is now clear that the levels of both hormone classes rise sharply during the perinatal period (for example see Polk, 1995; Baines et al. 2000). These data, when taken together with the present results, suggest that exposure to dexamethasone and T3 may underlie the increasing sensitivity to -adrenoceptor agonists that develops during the final stages of gestation (Walters & Olver, 1978; Brown et al. 1983).
Mechanism of hormone action
-Adrenoceptor agonists act by evoking cAMP synthesis and this allows such agonists to modulate the activity of adenine nucleotide-dependent protein kinases (PKA), enzymes that control many aspects of cellular physiology by phosphorylating specific protein targets (reviewed by Donowitz & Welsh, 1986; Levitzki, 1988). In fetal lambs, as well as blocking the maturation of the response to adrenaline, thyroidectomy prevents the developmental regulation of the response to a cell-permeant cAMP analogue, suggesting that the permissive effects of thyroid and glucocorticoid hormones are mediated at a site downstream of -adrenoceptor-mediated cAMP formation (Barker et al. 1988, 1990a). The present study tested this hypothesis by exploring the effects of isoprenaline upon the conductive properties of the apical plasma membrane. These experiments showed that isoprenaline had no effect upon GNa under hormone- and growth-factor-free conditions, but confirmed that this drug could regulate GNa in the dexamethasone and T3-treated cells. This failure to increase GNa under hormone- and growth-factor-free conditions cannot be attributed to a lack of ENaC expression, as mRNA encoding all three ENaC subunits is present in cells maintained in standard MDSF medium (Richard et al. 2002) and as basal GNa under these conditions is similar to the value recorded for hormone-treated cells. Moreover, withdrawing hormones/growth factors did not block isoprenaline-evoked cAMP synthesis, and so it is clear that functionally coupled -adrenoceptors are present in cells maintained under these conditions. The present study also showed that isoprenaline could rapidly increase GCl in both control and dexamethasone and T3-treated cells. Previous work has suggested that this response reflects increased activity of the cAMP-dependent anion channels formed by the cystic fibrosis transmembrane conductance regulator, the protein that is defective in cystic fibrosis (Welsh & Smith, 1993; Jiang et al. 1998; Collett et al. 2002). The fact that this rise in GCl is not associated with a discernible change in ISC implies that internal Cl- must be close to electrochemical equilibrium under the present conditions (see Collett et al. 2002). However, the most important point to emerge from these experiments is that they establish that isoprenaline can exert control over GCl in the absence of dexamethasone and T3, implying that these hormones are not necessary to allow isoprenaline to control PKA activity. The failure to evoke increases in GNa cannot, therefore, be attributed to the lack of functional -adrenoceptors or to impairment of their ability to activate adenylate cyclase/PKA. The present data thus provide the first verification of Barker's hypothesis (1988, 1990a) that dexamethasone and T3 are essential for the cAMP-mediated control of GNa in the fetal alveolar epithelium.
That the permissive effect of dexamethasone and T3 is specific to the regulation of GNa was initially surprising, as it is not immediately obvious how cAMP could activate anion channels without also regulating Na+ channels. However, Niisato et al. (1999) suggest that the two components of this response involve different mechanisms. cAMP-stimulated Na+ transport thus appears to be dependent upon enzymes that catalyse the phosphorylation of tyrosine residues (PTK), rather than the classical PKA-dependent pathway (reviewed by Donowitz & Welsh, 1986; Levitzki, 1988). The cAMP-dependent Cl- channels in these cells, however, seem to be controlled via cAMP/PKA (Jiang et al. 1998; Collett et al. 2002). There are also important physiological differences between the two responses as, instead of activating apical Na+ channels per se, cAMP-dependent agonists seem to increase GNa by stimulating an exocytotic process and thus allowing additional Na+ channels to be placed into this membrane (Ito et al. 1997). The rapid increases in GCl, on the other hand, appear to be due to cAMP-dependent control over anion channel activity (Jiang et al. 1998; Collett et al. 2002). Dexamethasone and T3 may thus act by facilitating an exocytotic process although the possible role of PTK in this response has yet to be explored (Ito et al. 1997; Niisato et al. 1999).
The present data thus show that thyroid and glucocorticoid hormones are crucial to the -adrenoceptor-mediated control over GNa in fetal distal lung epithelia and so, as well as inhibiting the secretion/synthesis of surfactant (Gross et al. 1984), our data predict that a lack of T3 would impair the clearance of lung liquid that normally occurs during the perinatal period. These effects may well explain why respiratory distress syndrome is abnormally prevalent amongst hypothyroid infants (see for example Redding & Pereira, 1974; Cuestas et al. 1976). Moreover, studies of fetal guinea-pigs have shown that premature delivery by Caesarean section attenuates the perinatal surge in circulating T3 levels (Baines et al. 2000) and, as this hormone seems to be essential for the proper control of GNa, this may explain why respiratory distress is more prevalent in babies delivered in this way (Fedrick & Butler, 1972).
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Acknowledgements
The authors thank the Wellcome Trust (Programme Grant no. 0548/Z/99/Z/JMW/CP/JF, Research Career Development Fellowship, S.K.I.) and Tenovus Scotland for the financial support that made this study possible, and are grateful to Helen Murphie and Elaine Waller for their skilled technical assistance.
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