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In pulmonary epithelia, β-adrenergic agonists regulate the membrane abundance of the epithelial sodium channel (ENaC) and, thereby, control the rate of transepithelial electrolyte absorption. This is a crucial regulatory mechanism for lung liquid clearance at birth and thereafter. This study investigated the influence of the gaseous signaling molecule hydrogen sulfide (H2S) on β-adrenergic agonist-regulated pulmonary sodium and liquid absorption. Application of the H2S-liberating molecule Na2S (50 μM) to the alveolar compartment of rat lungs in situ decreased baseline liquid absorption and abrogated the stimulation of liquid absorption by the β-adrenergic agonist terbutaline. There was no additional effect of Na2S over that of the ENaC inhibitor amiloride. In electrophysiological Ussing chamber experiments with native lung epithelia (Xenopus laevis), Na2S inhibited the stimulation of amiloride-sensitive current by terbutaline. β-adrenergic agonists generally increase ENaC abundance by cAMP formation and activation of PKA. Activation of this pathway by forskolin and 3-isobutyl-1-methylxanthine increased amiloride-sensitive currents in H441 pulmonary epithelial cells. This effect was inhibited by Na2S in a dose-dependent manner (5-50 μM). Na2S had no effect on cellular ATP concentration, cAMP formation, and activation of PKA. By contrast, Na2S prevented the cAMP-induced increase in ENaC activity in the apical membrane of H441 cells. H441 cells expressed the H2S-generating enzymes cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, and they produced H2S amounts within the employed concentration range. These data demonstrate that H2S prevents the stimulation of ENaC by cAMP/PKA and, thereby, inhibits the proabsorptive effect of β-adrenergic agonists on lung liquid clearance., (Copyright © 2015 the American Physiological Society.)

The epithelium of the developing lung displays an evolving liquid transport phenotype, in which Cl(-) secretion during fetal life is rapidly switched to Na(+) absorption perinatally. However, the mechanisms underlying the homeostasis of the thin layer of liquid lining the postnatal pulmonary epithelium remain elusive. In particular, it remains unclear whether the stimulated clearance of excess alveolar liquid is mediated via transepithelial Cl(-) transport. Our study is a pharmacological analysis with the aim of addressing this issue, which is of major physiological significance in cases of pulmonary oedema from any cause. We measured the rate of transepithelial liquid movement (J(v)) with (125)I-albumin, in the in situ perfused adult rat lung. Transepithelial Cl(-) transport was studied with the use of the Cl(-) channel inhibitor NPPB in the resting state and during stimulation with the β(2)-adrenergic agonist terbutaline. The study of J(v) in these conditions revealed the following findings: (1) there is net absorption of excess of alveolar liquid in the resting, unstimulated state, which is predominantly amiloride sensitive; (2) inhibition of Cl(-) transport with NPPB in the resting state results in a 1.6-fold increase in net absorption of alveolar liquid; and (3) the terbutaline-stimulated net absorption of the excess liquid is enhanced by 2.8-fold in the presence of NPPB. Our results are suggestive of the functional presence of secretory, but not absorptive, Cl(-) mechanisms and show that transepithelial Cl(-) transport is not part of the mechanism underlying lung liquid clearance in response to β-adrenergic stimulation.

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 μM L: -cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.

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.

The developing distal lung epithelium displays an evolving liquid transport phenotype, reflecting a changing and dynamic balance between Cl- ion secretion and Na+ ion absorption, which in turn reflects changing functional requirements. Thus in the fetus, Cl--driven liquid secretion predominates throughout gestation and generates a distending pressure to stretch the lung and stimulate growth. Increasing Na+ absorptive capacity develops toward term, anticipating the switch to an absorptive phenotype at birth and beyond. There is some empirical evidence of ligand-gated regulation of Cl- transport and of regulation via changes in the driving force for Cl- secretion. Epinephrine, O2, glucocorticoid, and thyroid hormones interact to stimulate Na+ absorption by increasing Na+ pump activity and apical Na+ conductance (GNa+) to bring about the switch from net secretion to net absorption as lung liquid is cleared from the lung at birth. Postnatally, the lung lumen contains a small Cl--based liquid secretion that generates a surface liquid layer, but the lung retains a large absorptive capacity to prevent alveolar flooding and clear edema fluid. This review explores the mechanisms underlying the functional development of the lung epithelium and draws upon evidence from classic integrative physiological studies combined with molecular physiology approaches.

This study was performed to determine whether the absorption of liquid from the lungs of postnatal sheep is dependent on pulmonary perfusion pressure, blood gases or blood flow. Relationships between perfusion pressure, rate of lung liquid absorption and perfusate PO2, PCO2 and pH were examined by linear regression analysis from in situ perfused lungs from sheep aged 6 weeks to 6 months. The airspaces of the lungs were filled with liquid containing an impermeant tracer, to allow measurement of the rate of liquid absorption. There was no significant relationship between the rate of lung liquid absorption and pulmonary blood flow (n = 36, r = -0.01, P > 0.1), pulmonary perfusion pressure (n = 36, r = 0.28, P > 0.05) or perfusate PO2, PCO2 or pH. No significant relationships were found between pulmonary blood flow and perfusate PO2, PCO2 or pH. There was no evidence to suggest that the absorption of liquid from the lungs of postnatal sheep is dependent on pulmonary blood flow, blood gases or perfusion pressure, within the limits studied, indicating that lung liquid absorption is dependent on the pulmonary epithelium and not on the pulmonary vasculature. The findings that lung liquid absorption continues in hypoxic environments and despite severe reductions in blood flow may be relevant to the field of transplant surgery.

1. Late gestation fetal sheep were chronically catheterised in utero to allow measurement of the rate of production of lung liquid (Jv) from 132-143 days gestation (term, 147 days), and to test the hypothesis that cyclic nucleotide gated cation channels mediate a component of fetal lung liquid absorption. 2. In eight experiments, 0.5 microg min-1 adrenaline caused a significant (P < 0.005) reduction in Jv from +18. 12 +/- 3.52 to -10.27 +/- 5.26 ml h-1. Dichlorobenzamil (a blocker of cyclic nucleotide gated cation channels) at 1.5 x 10-5 M did not significantly inhibit the adrenaline-induced lung liquid absorption (Jv dichlorobenzamil, -5.77 +/- 2.78 ml h-1; P > 0.1) when the data were grouped, but did exert a significant gestational effect (r = 0. 90, P < 0.01). Subsequent addition of 10-4 M amiloride (a blocker of epithelial sodium channels) abolished the adrenaline-induced absorption of lung liquid (mean Jv amiloride, +6.45 +/- 1.59 ml h-1; P < 0.01 relative to Jv adrenaline and P < 0.005 relative to Jv dichlorobenzamil). 3. In seven experiments, 0.5 microg min-1 adrenaline caused a significant (P < 0.0005) reduction in Jv from +18.95 +/- 2. 98 to -10.08 +/- 3.75 ml h-1. Amiloride (10-4 M) inhibited the adrenaline response (Jv amiloride, +5.46 +/- 1.09 ml h-1; P < 0.005). However, subsequent addition of 1.5 x 10-5 M dichlorobenzamil had no additive effect to that of amiloride (Jv dichlorobenzamil, +4.58 +/- 0.93 ml h-1; P > 0.1). 4. In six experiments, the cGMP analogue 8-Br-cGMP at 10-4 M caused a significant (P < 0.05) reduction in Jv from +15.20 +/- 2.81 to +11.63 +/- 1.71 ml h-1. Amiloride (10-4 M) did not block the effect of 8-Br-cGMP (Jv amiloride, +14.00 +/- 2.49 ml h-1; not significantly different from 8-Br-cGMP). Subsequent addition of 1.5 x 10-5 M dichlorobenzamil also did not block the effect of 8-Br-cGMP (Jv dichlorobenzamil, +11.37 +/- 1.22 ml h-1; not significantly different from either Jv amiloride or Jv 8-Br-cGMP). 5. We conclude that, in fetal sheep, neither adrenaline nor cGMP stimulate lung liquid absorption by actions on cyclic nucleotide gated cation channels, and that the effect of cGMP on fetal lung liquid secretion is minor and does not involve epithelial sodium channels. The effect of dichlorobenzamil, when given before amiloride, was probably due to an action on amiloride sensitive epithelial sodium channels.

1. Sheep lungs were artificially perfused in situ with warmed whole oxygenated sheep blood. The airspaces of the lungs were filled with liquid containing an impermeant tracer, to allow measurement of the rate of net transepithelial liquid movement under various conditions. 2. Dichlorobenzamil (1.5 x 10-5 M), a blocker of cyclic nucleotide-gated cation channels, inhibited the resting absorption of lung liquid in sheep aged 6 months (n = 5) (from -36.47 +/- 4.62 to -4.36 +/- 5.27 ml h-1, means +/- s.e.m.; P < 0.005, paired t test). Amiloride (10-4 M), a blocker of epithelial sodium channels, had no additive effect to that of dichlorobenzamil. 3. In the lungs of sheep aged 6 months (n = 4), amiloride (10-4 M) partially inhibited the resting absorption of liquid (from -35.21 +/- 8.57 to -11.05 +/- 4.91 ml h-1; P < 0.05, one-tailed paired t test), and dichlorobenzamil (1.5 x 10-5 M) exerted an additive effect to that of amiloride resulting in secretion at +6.29 +/- 3.05 ml h-1 (P < 0. 01, paired t test). 4. In the lungs of sheep aged 6 weeks (n = 3), amiloride (10-4 M) also inhibited the resting absorption of liquid (from -26.36 +/- 14.05 to -5.17 +/- 8.27 ml h-1; P < 0.05, one-tailed paired t test); however, dichlorobenzamil (1.5 x 10-5 M) did not exert an additive effect to that of amiloride. 5. In the lungs of sheep aged 6 months (n = 4), amiloride (10-4 M) partially inhibited the resting absorption of liquid (from -35.70 +/- 8.58 to -6.79 +/- 4.28 ml h-1; P < 0.05, paired t test), and pimozide (1.5 x 10-4 M), another blocker of cyclic nucleotide-gated cation channels, also exerted an additive effect to that of amiloride, resulting in secretion of lung liquid at +15.36 +/- 9.14 ml h-1 (P < 0.05, paired t test). 6. We conclude that cyclic nucleotide-gated cation channels mediate a component of lung liquid absorption in sheep aged 6 months (but not in sheep aged 6 weeks), and that a mechanism for lung liquid secretion (present in fetuses) is retained at 6 months of age.

Lung liquid (LL) is secreted into the fetal lung lumen, but it must be rapidly absorbed at birth to allow air breathing. In vitro studies have implicated oxygen as a possible factor causing the switch from secretion to absorption of lung liquid at birth. We developed a technique of oxygenating the fetal lung using liquid ventilation with haemoglobin (Hb) solutions in chronically catheterized fetal lambs (129-140 days gestation; term, 147 days). In some experiments 2,3-diphosphoglycerate (DPG) was added to increase oxygen delivery. LL secretion rate (Jv) was measured using an indicator dilution method. Eighteen fetuses were divided into four groups and ventilated with liquid under the following conditions: (i) Hb with oxygen, (ii) Hb without oxygen, (iii) Hb with DPG and oxygen and (iv) Hb and DPG without oxygen. There was a significant rise (2.6 mmHg, P < 0.02) in fetal arterial Po2 in group iii, but in none of the other groups. In the first 3 h of liquid ventilation there was no difference in Jv between the groups. In group i, during hours 4-6 of liquid ventilation, there was a significant rise in secretion rate from 2.25 +/- 0.88 to 3.74 +/- 0.85 ml h-1 kg-1 (P < 0.001). In group iii, when comparing Jv in the first 3 h of liquid ventilation with that in the following 3 h period of liquid ventilation, a strong trend towards reduction in secretion was observed, falling from 3.03 +/- 0.65 to 0.74 +/- 0.92 ml h-1 kg-1 (three of the four experiments showed a significant decrease in Jv in hours 4-6). These experiments indicate that oxygen delivered to the fetus using liquid ventilation with haemoglobin solutions leads to increased LL secretion when oxygen delivery is small, and suggest there is a decrease in secretion with greater oxygen delivery to the lung.

1. The lungs of two groups of lambs aged 0-2 weeks and 6-12 weeks were artificially perfused in situ with warmed and oxygenated sheep blood. The airspaces of the lungs were filled with liquid containing an impermeant tracer to allow estimation of net liquid movement across the pulmonary epithelium at rest and after administration of certain drugs. 2. Dibutyryl cAMP (dB-cAMP, 10-4 M) stimulated the rate of lung liquid (LL) absorption in the lungs of four neonatal sheep aged 9-12 days, from -1.43 +/- 0.2 to -2.75 +/- 0.3 ml h-1 (kg body wt)-1 (P < 0.05, comparison of regression lines by Student's t test), but had no effect in four juvenile sheep aged 6-12 weeks (P > 0.10). 3. Theophylline, a non-selective phosphodiesterase (PDE) inhibitor (5 x 10-4 M), increased LL absorption from a resting rate of -1.55 +/- 0.3 to -3.62 +/- 0.5 ml h-1 kg-1 in the lungs of four sheep aged 1-12 days and from -1.47 +/- 0.3 to -3.73 +/- 0.4 ml h-1 kg-1 in four sheep aged 6-12 weeks (P < 0.05, Student's paired t test). 4. The beta-adrenergic antagonist sotalol (10-4 M) reduced LL absorption rate from -1.47 +/- 0.1 to -1.22 +/- 0.1 ml h-1 kg-1 (P < 0.01) in the lungs of four sheep aged 4-13 days, while theophylline given after sotalol had no effect. In four sheep aged 6-12 weeks, sotalol had no effect on LL absorption rate, whereas theophylline given after sotalol increased LL absorption rate from -1.06 +/- 0.1 to -1.92 +/- 0.2 ml h-1 kg-1 (P < 0.05). 5. The A1/A2 purinergic receptor blocker 7-(beta-chloroethyl) theophylline (CET; given at 5 x 10-6 M and 10-4 M) had no effect on LL absorption rate in the lungs of four sheep aged 6-12 weeks, confirming that theophylline produced its effect of increasing LL absorption by inhibiting PDE hydrolytic activity. 6. The selective PDE IV (cAMP-specific) PDE inhibitor rolipram was given in the perfused lungs of seven sheep aged 6-12 weeks at doses between 10-8 and 10-4 M, increasing LL absorption rate at concentrations of 10-6 M and above; the half-maximal effective concentration was estimated to be 5.9 x 10-7 M. 7. Rolipram (10-5 M) increased LL absorption rate from -1.99 +/- 0.2 to -3.18 +/- 0.5 ml h-1 kg-1 in the perfused lungs of four sheep aged 6-11 days, and from -1.21 +/- 0.4 to -3.45 +/- 0.3 ml h-1 kg-1 in the perfused lungs of four sheep aged 6-12 weeks (P < 0.05). Sotalol (10-4 M) reduced LL absorption rate from -3.39 +/- 0.8 to -2. 18 +/- 0.4 ml h-1 kg-1 (P < 0.05) in four sheep aged 10-14 days, while rolipram given after sotalol had no effect. In four sheep aged 6-12 weeks, sotalol had no effect on resting LL absorption rate, whereas rolipram given after sotalol increased absorption rate from -1.27 +/- 0.1 to -2.02 +/- 0.6 ml h-1 kg-1 (P < 0.05). 8. We conclude that cAMP mediates a component of LL absorption postnatally, and that while beta-adrenergic stimulation was the sole source of endogenous cAMP in neonates, this was not the case in juveniles, in whom cAMP originated, at least in part, from other sources.

Previous methods of estimating the volume of epithelial lining liquid (ELL) in the air-filled lung may be said to have suffered from some theoretical and practical deficiencies. To estimate the ELL volume (VELL) more accurately, a known amount of artificial lung liquid (LL) containing the impermeant tracer 125I-albumin was instilled intraluminally in 56 in situ perfused postnatal sheep lungs (aged 36 h to 12 wk), and the rate of change of LL volume was measured by changes in the tracer concentration. Linear regression of LL volume against time allowed calculation of the VELL at the time of instillation; it was found to be 0.37 +/- 0.15 ml/kg body wt, which is equivalent to a film of 0.15 +/- 0.06 micron mean depth. The median ELL protein concentration was 36.8 mg/ml ELL, i.e., 0.60 times the plasma concentration, which agrees with previously published estimates and which is similar to the interstitial protein concentration. We conclude that the VELL is very small and that there is unlikely to be a protein osmotic gradient across the pulmonary epithelium.

1. The lungs of five fetal (133-140 days gestation) and thirty-four postnatal (2-240 days) sheep were artificially perfused in situ with warmed and oxygenated sheep blood. In postnatal animals the airspace of the lung was filled with liquid similar in composition to fetal lung liquid. In fetal and postnatal animals luminal liquid volume was measured by the impermeant tracer technique. 2. Under resting conditions the pulmonary epithelium of fetal animals secreted liquid at a mean (+/- S.E.M.) rate of 2.0 (+/- 0.4) ml (kg body weight)-1 h-1, those of postnantal animals absorbed liquid at -1.8 (+/- 0.2) ml (kg body weight)-1 h-1. 3. Addition of 2,4-dinitrophenol to achieve a concentration of 1.5 x 10(-3) M in the perfusing blood in postnatal animals caused complete cessation of liquid absorption. 4. Light and electron microscopic examination of the lung after periods of up to 6 h of artificial perfusion showed no evidence of epithelial damage. From 3 h onwards, liquid accumulation was evident in the perivascular spaces. 5. Addition of adrenaline to the perfusate in fetal animals caused absorption of liquid to occur at a mean rate of -2.9 (+/- 1.3) ml (kg body weight)-1 h-1. In postnatal animals adrenaline caused the rate of liquid absorption to increase from a mean rate of -1.4 (+/- 0.2) to -2.2 (+/- 0.3) ml (kg body weight)-1 h-1. 6. In the fetus addition of amiloride (0.8 x 10(-4) M) to the luminal fluid blocked adrenaline-induced liquid absorption and caused secretion to occur at 1.3 (+/- 0.3) ml (kg body weight)-1 h-1. 7. In postnatal animals the response to amiloride was age dependent. In newborn lambs (2-14 days) amiloride blocked liquid absorption and caused secretion of liquid to occur in seven out of eight animals at a mean rate of 0.9 (+/- 0.3) ml (kg body weight)-1 h-1 (n = 8). In older animals (15-240 days) the characteristic response to amiloride was slowing of the rate of liquid absorption (mean rate of absorption,-0.2 (+/- 0.09) ml (kg body weight)-1 h-1, n = 18) with liquid secretion being seen in only three of eighteen animals.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Thyroidectomy was performed on twelve fetal sheep between 111 and 115 days gestation. Measurement of fetal lung liquid secretion and absorption rates (Jv) were made at rest and during short (45 min) and long (5 h) infusions of adrenaline (0.5 micrograms/min) in a total of thirty-seven experiments, some in the absence of triiodothyronine (T3) and hydrocortisone and some at set times after the administration of the two hormones. 2. T3 was given either as an I.V. infusion (60 micrograms/24 h) or as a bolus of 30 micrograms; hydrocortisone was given as an infusion of 10 mg/24 h. Both hormones were administered together. 3. Before T3 and hydrocortisone were given short infusions of adrenaline had no effect on Jv but 4 h after exposure to the hormones secretion rate was reduced to near zero (Jv = -0.5 +/- 1.6 ml/h, n = 4) by adrenaline; after 24 h of hormone exposure, absorption of fetal lung liquid was produced by adrenaline (Jv = -3.6 +/- 2.2 ml/h, n = 4) which was even greater after 72 h, (Jv = -11.2 +/- 2.2 ml/h, n = 4). 4. During long infusions of adrenaline when T3 and hydrocortisone were given at the start of the experiment, an effect on lung liquid secretion was evident at 2 h and absorption was produced at 4 h (Jv = -4.2 +/- 2.5 ml/h, n = 3). The effect was significantly different from control long infusions of adrenaline performed the previous day in the absence of hormones. 5. After 24 or 48 h of stopping T3 and hydrocortisone administration, adrenaline no longer produced absorption of lung liquid, indicating that the effect of the two hormones was reversible within 24-48 h. 6. The protein synthesis inhibitor cycloheximide put into lung liquid (4 x 10(-5) to 3 x 10(-4) M) blunted the effect of the hormones at 4 h and prevented absorption of lung liquid at 24 h. Jv during adrenaline was -3.6 +/- 1.5 ml/h in control experiments but was +3.3 +/- 0.9 ml/h after cycloheximide, n = 4, P < 0.01. This indicated that the two hormones produced their effect through protein synthesis.

The influence of triiodothyronine and hydrocortisone on maturation of the response to epinephrine that leads to reabsorption of lung liquid was investigated in nine chronically catheterized fetal sheep. Experiments were performed on thyroidectomized fetal sheep at 116-120 d gestation, well before the reabsorptive response to epinephrine is normally seen. After i.v. administration of either triiodothyronine (60 micrograms/d) or hydrocortisone (10 mg/d) for 3 d (three fetuses in each case), all fetuses continued to secrete lung liquid during exposure to epinephrine (secretion rate = 5.9 +/- 3.2 mL/h in triiodothyronine-treated and 4.4 +/- 1.9 mL/h in hydrocortisone-treated fetuses). However, when the two hormones were administered together in the same doses to three fetuses, a striking reabsorptive response to epinephrine was seen (absorption rate = -12.3 +/- 3.6 mL/h), similar to that observed in the mature fetus. Induction of this capacity to reabsorb lung liquid may be of importance in the management of respiratory problems of the newborn infant.

1. Following thyroidectomy at 106-118 days fetal sheep were infused continuously with triiodothyronine (T3) from 110, 118, 125 or 131 days (n = 12) or with thyroxine (T4) from 118 days (n = 4) until the fetuses were delivered. Lung liquid secretion or absorption rates, heart rate, blood pressure and arterial blood gases were measured before and during 45 min periods of fetal infusions of adrenaline (n = 60) at 3-8 day intervals. The effects of T3 or T4 replacement on the response to adrenaline were compared with data previously obtained in groups of euthyroid (control) and thyroidectomized (Tx) fetuses. 2. Fetuses infused with T4 (50 micrograms/day) following thyroidectomy had plasma T4 and T3 concentrations in the normal fetal range. Fetal plasma T3 levels in fetuses infused with T3 (60 micrograms/day) were at or above the high end of the normal range for full-term fetuses. Those receiving 120 micrograms of T3 per day had levels equivalent to those normally seen in the postnatal T3 surge. 3. Normal maturation in the lung of the reabsorptive response of fetal lung liquid to adrenaline was seen in the fetuses infused with T3 or T4 from 118 days. A marginal advance in maturation was seen in fetuses infused with T3 from 110 days and a delay in maturation in those infused with T3 from 125 and 131 days.

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.

1. In fetal sheep at 113-120 days' gestation, thyroidectomy was performed and tracheal, arterial and venous catheters inserted. Following a recovery period experiments were performed from 120-145 days to measure changes in lung liquid secretion or its absorption in response to I.V. adrenaline infusion or to introduction of dibuteryl cyclic AMP into lung liquid. The results were compared with those previously obtained in non-thyroidectomized fetuses. 2. Plasma levels of thyroid hormones in non-thyroidectomized fetuses confirmed the pattern found by previous workers. In thyroidectomized fetuses the levels of thyroxine (T4), tri-iodothyronine (T3) and reverse T3 (rT3) were very low except in one fetus which showed biochemical evidence of thyroid regeneration towards the end of gestation. 3. In thyroidectomized fetuses the normal response to adrenaline infusion (diminution of reversal of lung liquid secretion) was profoundly suppressed and very little gestational maturation in this response took place, except in the one fetus with evidence of thyroid regeneration in which a normal reabsorptive response developed in late gestation. 4. In thyroidectomized fetuses, the normal response to dibuteryl cyclic AMP was greatly reduced and its increase with gestation which normally parallels that seen during adrenaline infusion did not take place.

1. Experiments were performed on exteriorized fetal lambs of between 69 days' gestation and term (147 days) in order to observe changes in lung volume and lung liquid secretion rate, and to delineate any alterations in solute permeability, ion transport and tight junction morphology in the maturing lung epithelium. Whilst it was technically possible to measure solute permeability as early as 69 days it was not feasible to apply the Ussing flux ratio technique before 84 days.2. Fetal lung liquid volume and secretion rate, when normalized for body weight, increase linearly with gestation, whereas tracheal volume expressed in the same manner remains constant.3. When expressed in terms of pore theory, epithelial permeability to small polar non-electrolytes does not change between 69 days and term (equivalent pore radius 0.66 nm and 0.64 nm respectively).4. In the immature fetus of 69-76 days, mean epithelial tight junction strand number is 8.3, whereas by the end of gestation it has fallen to 4.6.5. The transfer constants (min(-1)) for sodium and chloride movement in the direction lung liquid to plasma are, respectively, some 6 and 4 times greater at 84-87 days than at term.6. As in the mature fetus, the lung epithelium at 84-87 days actively transports chloride from plasma to lung lumen, albeit with a slightly reduced transport e.m.f. Sodium movement does not, at any gestational age, differ from the predictions for passive transfer.7. In lung liquid the concentrations of chloride and potassium increase and that of bicarbonate decreases during gestation, whilst that of sodium does not change. The rises in lung liquid chloride and potassium concentrations follow those in plasma, maintaining plasma/lung liquid ratios of 0.7 and 0.95 respectively. However, plasma bicarbonate remains constant and the plasma/lung liquid ratio for bicarbonate rises from 3 at 69-76 days to 20 near term as the lung liquid bicarbonate falls from 9.8 to under 2 m-mole kg(-1) H(2)O.8. Whereas lung liquid protein concentration remains constant and low at about 0.35 g l.(-1), plasma protein concentration rises from 23 g l.(-1) at 69-76 days to 43 g l.(-1) near term. During the same period arterial blood pressure doubles.

The integrity of epithelial tight junctions in foetal mammalian lungs is essential to maintain the unique ionic composition of lung liquid, and to prevent leakage of serum proteins into peripheral air spaces. In the present study the development of intercellular junctions of the lining epithelium of foetal lamb lungs during gestation was examined by light and electron microscopy. Both thin sections and freeze-fracture replicas were examined by electron microscopy. By 39 days of gestation, epithelial tight junctions consist of a minimum of 3.1 +/- 1.6 (s.D.) and a maximum of 5.8 +/- 2.0 discontinuous rows of particles and short segments of strands on P face ridges and in complementary E face grooves, while from 58 to 76 days they are composed of a network of 4.3 +/- 1.6 to 7.7 +/- 1.9 focally interrupted P face strands. Complementary replicas show that many of the discontinuities on the P face are due to separation of junctional particles on to the E face during fracturing, and not to an absence of junctional particles. From 76 days to term, epithelial tight junctions (exclusive of upper airway epithelium which was not examined) resemble those of adult lungs, and consist of a continuous network of 4.5 +/- 2.0 to 7.5 +/- 2.5 P face strands and complementary particle-free grooves. Permeability measurements, published elsewhere, indicate that the epithelium is functionally 'tight' from 69 days onwards. Tight junctions in peripheral air-space epithelium, therefore, are structurally continuous and functionally 'tight' early in foetal lung development, and form seals at one end of long, narrow intercellular spaces; these features may be important for coupled ion and water transport. When the bounding epithelial cells become flattened, these narrow intercellular spaces remain intact as a result of complex interdigitations of adjacent cell membranes. Desmosomes were present throughout gestation near the abluminal side of the tight junctions and occasionally near the base of the intercellular space. These junctions may serve to connect cells to each other at a time when tight junctions may be mechanically weak. In addition, gap junctions are associated with tight junctions from the glandular through the canalicular stages of lung development. They disappear by 120 days when the epithelial cells are differentiated.

Surface and electrokinetic properties of purified calf lung surfactant in various electrolyte solutions were determined. Surface properties were pH dependent in distilled water and the surfactant performed as a good lung surfactant only below pH 4. In more physiological media it was pH insensitive over the range 2-8.5. In distilled water at pH 6 its surface properties improved when NaCl was added up to 20 mM; above this concentration it had the surface properties required to stabilise alveoli. The surface properties of surfactant in distilled water were also restored by certain cations (Ca2+, Mg2+, Mn2+, Cd2+ and Ni2+) but not others (Na+, K+, La3+ and Fe3+) when added to an ionic strength of 5.6 mM. Cations that restored its surface activity also reduced the surface charge density on the surfactant particles. Aggregation of surfactant by various metal chlorides was studied by light scattering measurements and bore no relation to surface activity or the charge on the particles. Aggregation of surfactant particles by Ca2+, Cd2+ and Mn2+ was instantly reversed by addition of excess EGTA. The influence of electrolytes on the surface properties of lung surfactant is explained in terms of the electrostatic forces operating in the system.

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.

1. In the chronically catheterized sheep fetus between 122 and 143 days gestation the concentration of D-glucose in lung liquid was very low (usually less than 0.01 mM, the lower limit of detection of the analytical method) whereas the mean plasma concentration was 0.19 mM (S.E.M. 0.4, n = 13). 2. When the lung liquid concentration of D-glucose was raised to 1.67-5.00 mM, rapid uptake was observed until the concentration had fallen to its preceding low level. The uptake showed saturation kinetics (Vmax = 2.29-8.78 mumol/min, increasing with gestation; mean Km = 0.14 +/- 0.02 mM, n = 11, no change with gestation). This active uptake of glucose was blocked by phloridzin (10(-4) M). It was associated with a decrease in lung liquid secretion rate from which a change in net sodium flux could be inferred of an order suggesting one-to-one glucose-sodium co-transport. 3. Radiolabelled 3-O-methyl-D-glucose (3-O-meG) - a monosaccharide which is transported but not metabolized - was taken up rapidly from lung liquid and this rapid uptake was inhibited by D-glucose with 50% inhibition at 0.35 mM (+/- 0.08, n = 9). It was also inhibited by phloridzin (10(-4) M). 4. Radiolabelled 2-deoxy-D-glucose - a monosaccharide which is not a substrate for sodium-coupled transport - was taken up only very slowly from lung liquid; the rate of uptake was appropriate for passive diffusional transport and it was unaffected by the addition of D-glucose or phloridzin to lung liquid. 5. Intravenous infusion of D-glucose caused no detectable increase in the concentration of glucose in lung liquid unless phloridzin was added, when a slow increase was observed. 6. In two experiments with active transport blocked by phloridzin in lung liquid (10(-4) M), the rate of entry of labelled 3-O-meG from plasma to lung liquid was measured during intravenous infusion of this tracer for 29 and 23 h. The rates of entry were similar to the rate of efflux of the tracer from lung liquid when uptake was blocked by phloridzin or D-glucose, and similar to the rate expected for a metabolically inert tracer (i.e. it was some two orders of magnitude less than efflux from lung liquid in the absence of an inhibitor).(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenaline was infused intravenously at rates of 0.1-1.0 microgram/min into chronically catheterized fetal lambs (125-141 days gestation) to induce slowing of secretion or reabsorption of lung liquid. There was an electrical potential difference (p.d.) of -0.3 to -9.5 mV (mean -3.4 mV) between lung liquid and plasma (lung liquid negative) during control lung liquid secretion. In response to adrenaline infusion, the p.d. increased (lung lumen more negative) and this change was greatest (1.8 +/- 0.3 mV) in experiments in which reabsorption occurred. Measurements were made of bidirectional fluxes of Na+ and Cl- across the pulmonary epithelium during control lung liquid secretion and during adrenaline infusion. Adrenaline-induced reabsorption of lung liquid was associated with an increase in Na+ flux from lung lumen to plasma. Similar but smaller changes occurred when the adrenaline response was slowing of secretion. The difference between measured flux ratios and those predicted from the forces determining passive flux provided evidence for active transport of Cl- from plasma to lung lumen, as previously demonstrated by Olver & Strang (1974). When adrenaline was infused, there was evidence of active Na+ transport in the direction lung lumen to plasma and an associated decrease in active Cl- transport in the opposite direction. These changes were greatest when the response to adrenaline was reabsorption. Amiloride, when mixed into the lung liquid to give a calculated concentration of 10(-4) M, abolished the changes in p.d. and ion flux induced by adrenaline. In experiments using amiloride concentrations between 10(-8) and 10(-4) M it was shown that 50% inhibition of the reabsorptive response to adrenaline (KI) was induced by 4 X 10(-6) M-amiloride in the lung lumen. Thus adrenaline-induced slowing of secretion or reabsorption of lung liquid is mediated by active Na+ transport from lung lumen to plasma and depends on amiloride-inhibitable Na+ channels on the luminal surface of the pulmonary epithelium.

In the chronically catheterized fetal lamb, intravenous infusion of adrenaline at 0.5 microgram/min produced slowing of the secretion of lung liquid or its absorption, an effect which increased exponentially with advancing gestation. Between 120 and 130 days, the characteristic response was slowing of secretion, whereas after 130 days it was absorption. Stimulus-response curves, relating secretion or absorption rate to plasma adrenaline concentration, were obtained by infusing adrenaline into the fetus intravenously at rates between 0.1 and 1.0 microgram/min (0.55-5.5 nmol/min). These curves allowed estimation of the minimum concentration of adrenaline required to inhibit secretion [( Ai]) and this was found to decrease from 0.43 ng/ml. (2.35 nM) at 132-4 days' gestation to 0.029 ng/ml. (0.16 nM) at gestations above 140 days. During spontaneous labour there was a slowing of lung liquid secretion in the early stages followed by absorption during the last 50-150 min. The mean concentration of adrenaline in plasma increased from 0.087 ng/ml. (0.48 nM) in early labour to 6.86 ng/ml. (37.5 nM) in the last 50 min and to 7.17 ng/ml. (39.2 nM) in the early post-natal period. Mean noradrenaline levels at the same times were 1.71 ng/ml. (10.1 nM), 12.14 ng/ml. (71.8 nM) and 9.10 ng/ml. (53.9 nM). The relationship between the plasma adrenaline concentration and the rate of absorption during labour was similar to that found when adrenaline was infused at various rates into the non-labouring fetus of comparable gestational age. The upper airway of the fetus was shown to be capable of acting as a one-way valve allowing outflow but not inflow of liquid. Thus withdrawal of liquid at 5-20 ml./hr from the fetal trachea below the larynx caused closure of the upper airway and this result was obtained both when the recurrent laryngeal nerves were intact and when they were divided.